Frequently mutated genes/pathways and genomic instability as prevention targets in liver cancer
Frequently mutated genes/pathways and genomic instability as prevention targets in liver cancer
Chinthalapally V. Rao 1
Adam S. Asch 0
Hiroshi Y. Yamada 1
0 Stephenson Cancer Center, Department of Medicine, Hematology/Oncology Section, University of Oklahoma Health Sciences Center (OUHSC) , Oklahoma City, OK 73104 , USA
1 Center for Cancer Prevention and Drug Development, Department of Medicine, Hematology/Oncology Section, University of Oklahoma Health Sciences Center (OUHSC) , 975 NE 10th Street BRC1207, Oklahoma City, OK 73104 , USA
The incidence of liver cancer has increased in recent years. Worldwide, liver cancer is common: more than 60000 related deaths are estimated each year. In the USA, about 27 170 deaths due to liver cancer are estimated for 2016. Liver cancer is highly resistant to conventional chemotherapy and radiotherapy. For all stages combined, the 5-year survival rate is 15-17%, leaving much to be desired for liver cancer prevention and therapy. Heterogeneity, which can originate from genomic instability, is one reason for poor outcome. About 80-90% of liver cancers are hepatocellular carcinoma (HCC), and recent cancer genome sequencing studies have revealed frequently mutated genes in HCC. In this review, we discuss the cause of the tumor heterogeneity based on the functions of genes that are frequently mutated in HCC. We overview the functions of the genes that are most frequently mutated (e.g. TP53, CTNNB1, AXIN1, ARID1A and WWP1) that portray major pathways leading to HCC and identify the roles of these genes in preventing genomic instability. Notably, the pathway analysis suggested that oxidative stress management may be critical to prevent accumulation of DNA damage and further mutations. We propose that both chromosome instability (CIN) and microsatellite instability (MIN) are integral to the hepatic carcinogenesis process leading to heterogeneity in HCC and that the pathways leading to heterogeneity may be targeted for prognosis, prevention and treatment.
Liver cancer is common worldwide, especially in Southeast
Asia and sub-Saharan Africa, where hepatitis virus infection is
endemic. More than 600 000 deaths from liver cancer are
estimated worldwide each year. In contrast to an overall decreasing
trend in cancer deaths, the incidence of and deaths by liver
cancer in the USA have increased in recent years. Approximately
27 170 deaths are estimated for 2016 (
). About 90% of liver cancer
is hepatocellular carcinoma (HCC); the remaining 10% is
cholangiocarcinoma (bile duct cancer). Cirrhosis and nonalcoholic
fatty liver disease are prominent risk factors in the USA and are
often associated with alcohol abuse and obesity, respectively.
In the USA, the overall 5-year survival rate for patients
with liver cancer is 15–17% (
). Surgery is performed at early
stages, when the 5-year survival is 31%. However, less than
half of all patients with liver cancer are diagnosed at an early
stage. Later stage liver cancers are quite resistant to current
chemo- and radiotherapies. For patients with later stage
cancer, survival rates drop to 11% (regional) and 3% (metastatic).
Clinical trials with newer immunotherapies have shown
some signs of promise (
), but more time is needed to assess
the results on a larger scale. Thus, better therapies to treat
liver cancer, along with diagnostic, prognostic and preventive
measures for high-risk groups, such as those with hepatitis
virus, cirrhosis or nonalcoholic fatty liver disease, and those
who have been exposed to dietary aflatoxin, are desperately
Liver cancer is highly heterogeneous in terms of morphology,
genome composition and mutated genes (
). Not only is HCC
heterogeneous among patients, multiple HCCs that occurred in
a patient also showed significant heterogeneity (5) and signs of
tumor evolution (
). The heterogeneity of HCCs in a patient
may result in difficulty in clinic for designing customized
targeted approach for HCCs in such patients.
Since 90% of liver cancer is HCC and most studies have been
conducted with HCC, in this review, we primarily discuss the
results from HCC studies. Risk factors and known causes for HCC
include genetic factors (e.g. male gender, metabolic syndrome
and diabetes), carcinogens (e.g. aflatoxin), lifestyle/habitual
behaviors (e.g. tobacco smoking, alcohol abuse and obesity) and
biological factors/infection (e.g. hepatitis virus) (
to the risk factors depends on environment. Thus, there is much
variation among geological regions and time frame in liver
In Africa and Southeast Asia, hepatitis virus and aflatoxin
exposure are estimated to be accountable for 70–90% of HCC
). Hepatitis virus infection is a leading risk factor for HCC in
the USA as well. Among 1500 patients at United States Veterans
Administration hospitals who developed HCC from 2005 through
2010, the annual proportion of nonalcoholic
steatohepatitisrelated HCC (7.5–12.0%) and HCC cases associated with
hepatitis B (1.4–3.5%) remained relatively stable. The proportion of
HCC cases associated with HCV increased from 61.0% in 2005 to
74.9% in 2010, whereas HCC cases associated with alcohol abuse
alone decreased from 21.9% in 2005 to 15.7% in 2010 (Figure 1)
). Recent studies utilizing cancer deep sequencing suggest
that particular risk factors, such as hepatitis virus infection,
aflatoxin exposure or alcohol abuse, may have stronger links
to specific sets of pathways and types of HCC; from the
standpoint of affected pathways, there may be several HCC subtypes
These findings from several groups and HCCs in different
regions (especially in USA, Japan and China) indicated
complicating factors for genomics approaches. Statistical results
from conventional HCC tumor sequencing may be influenced
by the composition of the tumor sources. For example, deep
sequencing results of HCCs from regions where hepatitis virus
infection is endemic may portray a mutation and pathway
profile that is different from sequencing results from HCCs
resulting from nonalcoholic steatohepatitis/nonalcoholic fatty liver
disease. Hence, HCC heterogeneity must be investigated with
a view toward identifying both etiological cause and affected
pathways in the resulting HCC. This is important because,
in the clinic, high heterogeneity means lack of consistency
in therapeutic outcome. Targeted therapy, like sorafenib,
may prove ineffective due to its limited range of
applicability. Therefore, molecular tumor typing should be refined and
guidelines for personalized cancer therapy approach should
be established with molecular markers to improve therapy
Figure 1. Risk factors and paths to liver cancer. HCC count as 90% of all liver cancers, whereas remaining 10% as cholangiocarcinoma (cancer in bile duct). Major known
risk factors can be categorized as genetic factors, carcinogens, lifestyle/habitual behaviors and biological factors/infection. Although significant numbers of HCC are
related to lifestyle, HBV/HCV-positive HCC remain most common (55% of HCC) in the USA [based on Mittal et al. (
)]. Human liver cancer genome sequencing studies
have begun to uncover relationship between risk factor/probable causes and mutated genes. Profiling of gene mutations according to the risk factors should follow.
Major types of genomic instability: (CIN and MIN)
Many liver cancers show high degrees of genomic instability.
Genomic instability is roughly categorized as mitotic
errormediated chromosome instability (CIN) and DNA metabolism
defect-mediated microsatellite instability (MIN). Although CIN
and MIN can coexist, in most cancers, one form is dominant. In
HCC, CIN is prevalent (
). CIN is generally linked to poorer
prognosis than MIN. One reason for this poor prognosis is that
genomic instability can serve as a mutator, increasing the rate
of mutation that may permit the cancer to adapt to challenges
from therapies (25). CIN in particular can cause large-scale
genomic alterations, such as chromothripsis or copy number
variations, and is hazardous to genomic integrity (
Mouse models for CIN and cancer, including HCC
As stated in the previous section, clinical samples can be affected
by etiological fluctuations. As a complementary approach,
genetically defined animal models for genomic instability can
provide more conclusive proof in terms of biology. In the past
years, carcinogenesis studies with CIN mouse models emerged
). Tumor developments were observed in the CIN models,
and the models were investigated with more interest in certain
organs that proved to be prone to cancer (i.e. lung and liver) or
whose cancer carries high CIN (i.e. colon) thus far. With these
models, several new findings about CIN on carcinogenesis and
on cancer have emerged. In an experiment with a CIN model,
mitotic checkpoint component Mad2-overexpressing mice, lung
tumor recurrence was enhanced with Mad2 overexpression,
indicating that the presence of high CIN can increase
recurrence and, hence, poor prognosis (
). In an experiment with
another CIN mouse model, regulator of chromosome cohesion
and centrosome integrity Sgo1 haploinsufficient mice,
spontaneous HCC were observed, and with hepatic and colonic
carcinogen Azoxymethane treatments, HCC developed in 7 of 10
Sgo1 mice, whereas of the 9 control mice none developed HCC,
showing a significant increase in proneness to HCC (P < 0.05)
), also supporting the notion of pro-carcinogenic effect of CIN.
However, another important notion that emerged is that CIN
can also work against carcinogenesis, by increasing cell death
). In an experiment with aged cenpe−/+ mice, spontane
ous HCC development rate was lower, which was interpreted as
a result of increased cell death in the liver (facilitated by CIN and
genomic damage) in the model (
). It is theorized that
carcinogenesis or tumor suppression with CIN is a result of a balance
between CIN-mediated DNA damage/mutation (with low-grade
aneuploidy) and CIN-facilitated cell death (with high-grade
In addition, CIN may influence tumor outcome with other
routes than genomic instability and countering cell death. In the
Sgo1 mice, colonic transcriptome was altered in pro-carcinogenic
manners, including activation of oncogenic signaling (e.g.
Wnt, PPAR and insulin), reduced oxidative stress response and
reduced immune function (
). Transcriptomic changes were
observed in other organs, including the lung and liver, where
cancer proneness in the model was found, and the
transcriptome alterations showed similar characteristics to the colon to
some extent (lung: 38, liver: C.V. Rao et al., in preparation). The
transcriptome results suggest that introducing genomic
instability in the tissue may create an environment that is
pro-carcinogenic and pro-cancer survival (e.g. activation of oncogenic
pathway(s), reduced oxidative stress response and reduced
immune function) (
The results from the CIN mouse models showed some
similarities to those in human HCC. Wang et al. identified human
Sgo1 protein accumulation in human HCC and proposed Sgo1
as a potential target for therapeutic intervention (
). The HCC
developed in Sgo1−/+ mouse model also showed Sgo1 accu
mulation. Moreover, the mouse model study indicated that
DNA-damaging reagents induced accumulation of Sgo1 in the
nucleus, suggesting that the Sgo1 protein may be accumulated
in nucleus in HCC with a possible functional role of DNA
damage response or repair (
Other paths from genomic instability to carcinogenesis
Further, genomic instability can generate aneuploid cells.
Aneuploidy affects the transcriptome and proteome, leading
to proteotoxic stress and activation of the endoplasmic
reticulum stress response. Therefore, aneuploidy can modulate
characteristics of the cells and the microenvironment (
with genomic damage can go senescent, which may contribute
to the deterioration of surrounding tissue functions through
senescence-associated secretory phenotype (SASP) (
can occur in human liver. The hepatocyte SASP included
characteristic factors such as interleukin (IL)-8 and IL-6, as well as
novel components such as SAA4, IL-32 and fibrinogen (
switch in secretome is regulated by Notch 1 in mice hepatocytes.
Notch signaling seems to modulate SASP composition in senes
cent hepatocytes, controlling the immune reaction in the liver
and thereby negatively regulating the elimination of senescent
hepatocytes, at least in part through suppressing T-lymphocyte
recruitment to the liver (
). SASP is involved not only in aging
but in HCC therapeutic interventions such as chemotherapy and
radiotherapy as well. For example, SASP is shown to be induced
by radiation treatments on rat liverin vivo (
drug treatment can cause SASP in liver, and attenuation of SASP
can promote HCC progression, thus SASP can play a suppressive
role on HCC. A variant of histone H2A, macroH2A1, is a marker of
senescence-associated heterochromatic foci. Chemotherapeutic
and DNA-demethylating agent 5-aza-deoxycytidine (5-aza-dC)
induced senescence through epigenetic regulation by
macroH2A1 and DNA methylation, and depletion of macroH2A1
amplified the antiproliferative effects of 5-aza-deoxycytidine
in HCC cells (
). Thus, genomic instability may lead to tissue
dysfunction and carcinogenesis through many emerging paths.
HBV or HCV infection can cause genomic instability in the liver
Genomic instability has a variety of causes, including mutation
or epigenetic misregulation in genes that maintain genomic
integrity, for example, CIN- or MIN-related genes, virus or
retrotransposon, genotoxic agents and radiation or thermal
stress. Reduced removal of cells with genomic instability due
to immune dysfunction, cell death defect or issues with
tissue homeostasis can also increase genomic instability (
most relevant genomic instability-inducing agents in the liver
are hepatitis virus B (HBV) and hepatitis virus C (HCV) (
and HCV can induce genomic instability in at least three ways:
(i) by interfering with mitotic regulator proteins directly, (ii)
by integrating the viral genome into various sites in the host
genome and (iii) by causing lingering inflammation in the liver.
For (i), HBV viral protein HBX can bind directly to mitotic spindle
checkpoint protein BubR1 (
). Overexpressed HCV viral protein
NS5A can disturb mitotic processes and cause genomic
). Interfering with these mitotic regulator proteins can
cause CIN-type genomic instability. For (ii), HBV encodes the
regulatory HBx protein, the primary role of which is to promote
transcription of the viral genome, and the viral genome then
persists as an extrachromosomal DNA circle in infected cells (
Viral genome integration occurs at various sites in the genome,
but when integration occurs at a locus where a
carcinogenesisrelevant gene lies and interferes with the gene’s function, the
effect may later be detected. For (iii), genomic instability can
result in cell death. Perhaps due to an increase in dying cells,
inflammation markers and inflammatory cytokines like IL6 tend
to be high in tissues with genomic instability. In Sgo1 CIN model
mice, hepatic IL6 expression and serum HCC marker
alphafetoprotein amount increased, consistent with the notion that
genomic instability alone can lead to sterile tissue inflammation
in liver and other organs (
Are the genes and pathways that are frequently mutated in HCC involved in genomic instability?
Earlier, we examined the functions of genes that are frequently
mutated in colon cancer in order to elucidate why CIN is so prevalent
in colon cancer (80–90% of occurrence) and identified the
progressive nature of CIN in colon cancer. That is, many of the frequently
mutated genes in colon cancer (e.g. APC, TP53 and FBXW7)
maintain mitotic fidelity, thereby preventing CIN. As mutations
accumulate, degree of CIN progressively increases (
). Because HCC also
exhibits a high degree of heterogeneity and genomic instability that
may or may not be attributed solely to hepatitis virus infection, we
hypothesized that the genes frequently mutated in HCC may also
have functions of maintaining genomic stability and examined the
functions of the frequently mutated genes and pathways from the
standpoint of their relations to genomic instability.
Contemporary omics approaches and tumor
mass-sequencing have identified various gene mutations, genome alterations
and epigenetic misregulations in several tumors, including
those in liver cancer (
). Additionally, technological
advances that enabled omics approaches with smaller
number of samples (e.g. single-cell sequencing) led to mutational
linage analyses in one tumor or among multiple tumors (
Demonstrating cancer development (or mutational ‘evolution’
during the cancer development) and increasing degree of
heterogeneity has become possible through phylogenic analysis (
A characteristic trait in genetic/genomic alterations in liver
cancer is the broad spectrum of apparent mutations and limited
number of encompassing mutations. For example, 88–100% of
pancreatic cancers show k-ras mutation, which strongly
suggests that k-ras mutation may serve as a critical ‘bottle neck’
initiator in pancreatic carcinogenesis. In contrast, except for
telomerase reverse transcriptase (TERT) promoter (60%
mutation rate), TP53/p53 (35–50% mutation rate) and CTNNB1/
beta-catenin (19–40%), mutation rates for the most frequently
mutated genes in HCC are relatively low [e.g. AXIN1 (13%),
ARID1A (12%) and WWP1 (9%)]. Although there is some
variation among studies, most of the ‘most frequently mutated
genes’ were mutated in <10% of HCC samples examined
). In addition, microRNA expressions and epigenetic
factors, such as EZH2, were determined to play a role in HCC,
adding more complexity to HCC tumor heterogeneity (50).
In the following sections, we will discuss genes that are
frequently mutated in human HCC, identified by recent genomic
analyses based on next-generation sequencing (NGS) . The
genes are selected based on higher mutation rates in genome
sequencing data [compiled from references (
most recent review, see ref. (14)] and presented in order from
higher mutation rate to lower rate. The overview of the genes
that are frequently mutated in human HCC will focus on most
heavily mutated genes, with cutoff at approximately 2–3%
mutation rate (
By collating the genes and identifying the pathways to which
they belong, several signaling pathways that may contribute to
HCC development were identified: (i) oncogenic pathways (Wnt,
Smad and EGFR), (ii) DNA damage checkpoint and repair
pathway, (iii) oxidative stress response pathway, (iv) cell cycle
pathway and (v) immune function pathways (Figure 2).
Then, we investigated how the pathways may be involved
in maintaining genomic integrity. Some mutations could cause
genomic instability by itself (e.g. TP53, Wnt signaling genes
including CTNNB1, AXIN1; chromatin modulators including
ARID1, MLL2/KMT2D; and DNA damage checkpoint or repair genes
including ATM). In addition, there are pathways whose
simultaneous loss can cause additive or synergistic damage to genome
integrity. Namely, when the DNA damage checkpoint and repair
and oxidative stress response pathways are simultaneously
impaired, extensive DNA damage or proneness to DNA damage
can occur. Unrepaired or misrepaired DNA double-strand breaks
lead to the formation of chromosome aberrations. Replication
) and inappropriate activation of the DNA damage repair
pathway during mitosis (
) have also been shown to cause CIN.
Genes that are frequently mutated in liver cancer: their role and involvement in genomic instability
In this section, we will discuss genes that are frequently
mutated in HCC individually. The genes are selected based on
higher mutation rates in genome sequencing data [compiled
from references (
)] and presented in the order from
higher mutation rate to lower rate. Genes with <2% mutation
rate are not discussed in this review.
TERT promoter (gain of function, 60%)
This gene (including the promoter region) has a mutation rate of
60% in HCC. It is associated with increased telomerase
expression/gain of function. Mutations in TERT promotor created an
E-twenty-six family/T-cell factor transcription factor binding
site and induced telomerase promoter activity and TERT
transcription. They were among those most frequently found in
liver cancer, leading to overactive telomerase (
of telomerase and telomere shortening is associated with DNA
double-strand breaks, genomic instability and senescence. In
contrast, overexpression of telomerase elongates telomeres and
aids immortalizing cells, benefiting cancer cell survival.
TP53 (loss of function, 35–50%)
TP53 encodes p53, one of most investigated tumor suppressors
with multiple functions. Wild-type p53 suppresses the
accumulation of aneuploid cells (
) and plays a major role in
maintaining genomic stability. Loss of p53 function can permit survival
or propagation of aneuploid cells, especially when combined
with mitotic error-generating mutations, such as spindle
checkpoint defects and/or Rb defect (
). p53 mutation can
produce centrosome amplification and CIN (58). Laurent-Puig et al.
reported that high CIN in HCC is associated with p53 mutation
and hepatitis virus infection, whereas low CIN is associated
with beta-catenin mutation (
). The association is consistent
with the notion that p53 mutation can lead to CIN. However, p53
mutation alone may not be a strong driver of CIN, and additional
mutation(s) may be needed to develop cancer.
CTNNB1 (gain of function, 20–40%)
Beta-catenin (gene: CTNNB1) is the effector of Wnt signaling.
The C-terminus region is found to be heavily mutated in
cancers including HCC. Since the amino terminus is involved in
degradation of β-catenin, the deletion likely results in its stabi
lization, leading to activation of Wnt signaling (gain of function)
). Its gain of function activates various pro-growth genes as
a transcription factor. Overactive Wnt signaling is observed in a
variety of cancers, and Wnt signaling is considered oncogenic.
β-Catenin–induced T-cell lymphoma by promoting genomic
instability. Activated beta-catenin altered double-strand break
repair and increased survival of thymocytes with damaged DNA,
which promoted genomic instability and formation of T-cell
). Inhibitors of a Wnt signaling component glycogen
synthase kinase 3 (GSK-3) induced chromosome instability (
Overall, these reports suggest that beta-catenin and Wnt
signaling activation can cause genomic instability, especially when
combined with an increase in DNA damage or mismatch repair
defects, which often occur in the process of HCC development.
AXIN1 (Loss of function, mutation rate, 9–13%)
AXIN1 is also a component of Wnt signaling, and the protein
interacts with other Wnt signaling proteins, adenomatosis
polyposis coli (APC), β-catenin (CTNNB1), GSK3β, protein
phosphatase 2 and itself. However, despite belonging to the same
pathway, genetic alterations in CTNNB1 and AXIN1 are mutually
exclusive, possibly because they carry opposite roles in terms
of pathway activation (
). AXIN1 is a negative regulator of Wnt
signaling, and AXIN1 loss of function activates Wnt signaling.
Ectopic expression of Axin1 paralog Axin2 or its up-regulation
through small interfering RNA-mediated knockdown of
adenomatosis polyposis coli, led to CIN in chromosomally stable colon
cancer cells, indicating the capability of Wnt signaling to induce
genomic instability (
LAMA2 (loss of function, mutation rate, 5–12%)
LAMA2 gene encodes Laminin subunit alpha 2, a major component
of the muscle basement membrane, and mutations in this gene
have been identified as the cause of congenital merosin-deficient
muscular dystrophy, an autosomal recessive disease typically
presenting as a severe, early onset congenital muscular dystrophy
). Laminin-α2 is expressed in skeletal muscle myoblasts and
myotubes where it promotes the survival of satellite cells as well
as myoblast fusion and myotube formation (
). The mutation
in HCC was identified in a 2014 study with a high frequency (6
of 42, 12%), and retrospective analysis in other HCC studies also
reported 6% and 5% mutation rates (
). LAMA2 is frequently
mutated in other cancers, including lung adenocarcinoma (11%),
lung squamous cell carcinoma (13%), uterine corpus
endometrioid carcinoma (13%) and head and neck squamous cell carcinoma
). LAMA2 is suggested to play a role as a tumor
). LAMA2−/− mice were viable, but showed signs of muscle
dystrophy and died by 5 weeks of age (
). LAMA2−/− cells showed
a higher percentage of mononucleic population (
). However, its
link to genomic instability has not been demonstrated.
ARID1A and ARID2 (ARID1A: loss of function, 12%. ARID2: loss of function, 7%)
ARID1A (AT-rich interactive domain-containing protein 1, aka
BAF250a) and ARID2/ARID1B (BAF200) are members of the
switch (SWI)/sucrose non-fermenting (SNF) family and are
thought to regulate transcription of certain genes by altering the
chromatin structure around those genes. As the loss of ARID1A
increases cancer incidence in several organs (e.g. breast,
gatrointestinal tract and ovary) and is a marker for poor prognosis, it
is considered to be a tumor suppressor. ARID1A function is also
implicated in regulation of mismatch repair, as its loss is
associated with MIN. For example, in microsatellite unstable
colorectal cancer, frequent inactivating mutations in ARID1A, ARID1B,
ARID2 and ARID4A were observed (
Arid1a deficiency initiated steatohepatitis and HCC in mice (
Reduced expression of ARID1A is associated with poor
prognosis and promotes HCC metastases. Consistently, siRNA-targeted
loss of function of SWI/SNF chromatin remodeling genes led to
genomic instability with increased mutation rates in human
lung cancer cells (
). In Japanese study, inactivating mutations
were significantly enriched in non-HBV and non-HCV patients,
suggesting a key tumor suppressor function of SWI/SNF
complexes in metabolic/toxic rather than virus-related HCC (
WWP1 [9%: functional link unclear (possibly, gain of function)]
WWP1 (WW domain-containing Protein 1/NEDD4-like E3
ubiquitin-protein ligase) is a E3 ubiquitin ligase that can target
multiple substrates, including KLF2, TGF beta, tumor suppressor
smad4, p27, ErbB4, ErbB2 and EGFR. Involvement of WWP1 in
regulations of oncogenic signaling with Smad4 and EGFR appears
particularly relevant in hepatic carcinogenesis. Overexpression
of WWP1 promoted tumorigenesis and predicted unfavorable
prognosis in patients with HCC (
Some WWP1 targets are involved in genomic instability. For
example, Smad4 loss in mice caused spontaneous head and
neck cancer with increased genomic instability and
). p27kip1 Deficiency impaired G2/M arrest in response to
DNA damage, leading to an increase in genetic instability (
Therefore, WWP1 misregulation, especially gain of function, can
indirectly cause genomic instability.
RPS6KA3 (90 kDa, polypeptide 3/RSK2; likely loss of function, mutation rate, 8%)
This gene encodes a member of the ribosomal S6 kinase (RSK)
family of serine/threonine kinases. Mutations in RPS6KA3 cause
Coffin–Lowry syndrome, a rare X-linked dominant disorder
characterized by intellectual disability, craniofacial
abnormalities, short stature, tapering fingers, hypotonia and skeletal
). RSK2 affects p53-mediated downstream cellular
events in response to DNA damage. RSK2 knockout relieves cell
cycle arrest at the G2/M phase and an increased number of γH2AX
foci, which are associated with defects in DNA repair. Therefore,
RSK2 plays an important role in the DNA damage pathway that
maintains genomic stability by mediating cell cycle progression
and DNA repair (
ATM (Likely loss of function, mutation rate, 7%)
Ataxia telangiectasia mutated (ATM) is a kinase involved in DNA
damage checkpoint and repair. Loss of ATM reduced
hepatocellular apoptosis and fibrosis in a high fat-fed mouse model of
nonalcoholic fatty liver disease, indicating that activation of
ATM in response to oxidative stress plays a role in development
of hepatic fibrosis (
Further work with mouse models indicated that ATM plays
a role in suppressing intestinal carcinogenesis when combined
with apc; therefore, ATM is a tumor suppressor. Defects in ATM
inactivate p53BP1-MDC1-nbs-mediated DNA checkpoint and
repair and sensitize cells to DNA damage and oxidative stress.
ATM and p21 cooperate to suppress aneuploidy and subsequent
tumor development (
). ATM defects alone may not be strong
drivers for genomic instability but with other mutations (e.g.
p21), genomic instability can manifest. Dramatically increased
CIN was observed in brains of human ATM patients (
). As in
these examples, ATM defects can make cells prone to increased
MIN and CIN.
CDKN2A [loss of function (most cases), mutation rate, 6–30%]
CDKN2A encodes p16INK4A, which is a CDK inhibitor, a senescent
protein, and a tumor suppressor. p16 suppresses cell cycle
progression and inducing senescence. siRNA-mediated inhibition of
p16 led to an increase in oxidative stress, that is, ROS and
oxidative (8-oxoguanine) DNA damage (
), suggesting that loss of p16
can worsen genomic instability if combined with DNA damage
checkpoint and repair defects. Consistently, p16INK4A-silencing
augments DNA damage-induced apoptosis in cervical cancer
). Oxidative stress can cause genomic instability by both
CIN and MIN.
MLL2/KMT2D (loss of function, mutation rate, 6%)
KMT2D encodes lysine methyltransferase 2D and functions as a
histone methyltransferase. Truncating mutations in the KMT2D
gene have been identified in people with Kabuki syndrome, a
disorder characterized by distinctive facial features, intellectual
disability and abnormalities affecting other parts of the body
). KMT2D mutation can be causal to B-cell leukemia, thus
KMT2D is considered a tumor suppressor 8(
evidence supports that KMT2D is involved in the regulation of gene
expression. In B cells, KMT2D sustains gene expression program
that represses B-cell lymphoma development (
of KMT2D perturbs germinal center B-cell development and
promotes lymphomagenesis (
). Kantidakis et al. identified
KMT2D as an interacting protein with RECQL5, which
associates with RNAPII subunits and several transcription-related
factors and also with genomic instability. The authors
suspected involvement of KMT2D in transcription. Although
showing only small transcriptional changes, mouse cells in which
KMT2D gene deletion can be induced, along with human cells
with KMT2D knockout, display elevated levels of sister
chromatid exchange, gross chromosomal aberrations, 53BP1 foci and
micronuclei. KMT2D mutation gives rise to significant genomic
instability in areas overlapping with early replicating fragile
sites. This may be explained by the involvement of KMT2D in
transcript elongation and specifically in mediating
elongationassociated histone H3K4 methylation ( 82). Thus, KMT2D
mutation can cause transcriptional stress, creation of fragile sites and
NFE2L2 (loss of function, mutation rate, 5%)
Nuclear factor [erythroid-derived 2]-like 2 (NFE2L2), or Nrf2, is a
transcription factor with basic leucine zipper (bZIP). Nrf2
regulates the expression of antioxidant proteins that protect against
oxidative damage triggered by injury and inflammation. Several
drugs that stimulate the NFE2L2 pathway are being studied for
the treatment of diseases that are caused by oxidative stress.
In myelofibrosis and related blood cancers, Nrf2 expression is
downregulated. Nrf2 knockout enhances intestinal
tumorigenesis in Apc (min/+) mice due to attenuation of the antioxida
tive stress pathway, which potentiates inflammation (
findings illustrate the importance of Nrf2-mediated oxidative
stress management in preventing carcinogenesis. Although
evidence for Nrf2 defects directly causing genomic instability has
not been found, Nrf2 defects may make cells prone to
oxidative stress-mediated DNA damage. If combined with DNA
damage checkpoint and repair defects, such as ATM or TP53 defect,
genomic instability would follow under oxidative challenge.
ERBB receptor feedback inhibitor 1/MIG6 [loss of function (which activates EGFR pathway), mutation rate, 5%]
MIG6 is a multifunctional adaptor protein and binds, in
particular, to the epidermal growth factor receptor (EGFR) kinase domain
and inhibits signaling. The EGRF-mitogen-inducible gene 6 (MIG6)
signaling axis plays a role in lung cancer (
). Mig6 is a sensor of
EGFR inactivation that directly activates c-Abl to induce apoptosis
during epithelial homeostasis. Hepatocyte growth factor (HGF)/
Met signaling can also be inhibited by Mig6 in cells of hepatic
origin and neurons, affecting cell migration, in a
Cdc42/Racdependent manner. Thus, MIG6 is involved in the inhibition of the
EGFR and HGF signaling pathways, and loss of function in MIG6
activates these oncogenic pathways. When MIG6 gene expres
sions were compared in HCC and hepatoblastomas,
hepatoblastomas showed 3.88-fold higher expression. As hepatoblastomas
tend to harbor fewer genomic alterations, low expression of MIG6
may be involved in genomic instability. Although circumstantial
evidence exists, a direct link between misregulation of MIG6-EGFR
or HGF and genomic instability in the liver remains to be found.
ZIC3 (mutation rate, 5%)
ZIC3 is a member of the zinc finger of the cerebellum (ZIC)
protein family and is a part of the transcription factor complexes
involved in development, especially of the heart, and
morphogenesis. During gastrulation and neurulation, Zic3 acts by
binding the distal regulatory regions associated with control of gene
transcription in the Nodal and Wnt signaling pathways. Zic3 is
also involved in regulation of stem cells and
generation/conversion to pluripotent progenitor cells. In a zebrafish model,
Zic3 was implicated in the regulation of BMP, Wnt and FGF (
Consistent with the ability of Zic3 to control multiple
development or oncogenic pathways, mutations and expression
abnormalities of Zic3 have been found in other cancers, including
lung, blood and brain cancer (
ALB (serum albumin; functional link unclear, mutation rate, 5%)
Serum albumin is synthesized in the liver and is involved in
the transport of various biomolecules, including hormones,
fatty acids, unconjugated bilirubin, drugs and ions. In anin vitro
model with rat primary neuronal cultured cells, serum
albuminattenuated DNA damage, and the effect was suggested to be due
to antioxidant properties through catalase activation. However,
analbuminemia alone did not significantly influence
hepatocarcinogenesis when F344 rats were compared with a congenic line
carrying the analbuminemic mutation (
). Thus, alb mutation
may indirectly contribute to oxidative stress management and
affect the microenvironment.
MLL3/KMT2C (loss of function, mutation rate, 4%)
This gene is a member of the myeloid/lymphoid or
mixed-lineage leukemia (MLL) family and encodes a nuclear protein with
an AT hook DNA-binding domain, a DHHC-type zinc finger, six
PHD-type zinc fingers, a SET domain, a post-SET domain, and a
RING-type zinc finger. MLL3 is a member of the ASC-2/NCOA6
histone–methyltransferase complex (ASCOM) and is involved in
transcriptional coactivation and epigenetic regulation. MLL3 can
serve as a p53 coactivator and functions as tumor suppressor
). Mutations in MLL3 were found in various tumors,
including bladder, colon, lung and pancreas tumors. Low expression
of MLL3 was linked with a low survival rate compared with
positive MLL3 expression in patients with gastric cancer. MLL3
may function like aforementioned MLL2/KMT2D and may be
involved in chromatin positioning and genomic instability (
Additionally, MLL3 may affect chemoresistance. Loss of the
MLL3/4 complex protein, PTIP, protects Brca1/2-deficient cells
from DNA damage and rescues the lethality of Brca2-deficient
embryonic stem cells (
IRF2 (likely loss of function, mutation rate, 4%)
Interferon regulatory factor 2 (IRF2) is thought to be an
oncoprotein. IRF2 competitively inhibits the IRF1-mediated
transcriptional activation of interferons alpha and beta and
presumably other genes that employ IRF1 for transcription act-i
vation. Although siRNA-mediated knockdown of IRF2 in
leukemic TF-1 cells resulted in growth inhibition associated with G2/M
arrest as well as induction of polyploidy, differentiation and
), forced expression of the IRF2 caused polyploidy
and cell death in FDC-P1 myeloid hematopoietic progenitor cells
). Thus, IRF2 misregulation may result in cell cycle
miscoordination and polyploidy, which may promote genomic instability
BAZ2B (Functional link unclear, mutation rate, 3%)
BAZ2B (bromodomain adjacent to zinc finger domain 2B) is a
bromodomain-containing protein whose function remains to be
determined. Bromodomain proteins are thought to interact with
acetylated lysine and are found in many chromatin remodeling
UBR3 (functional link unclear, mutation rate, 2%)
Ubr3 (ubiquitin protein ligase E3 component N-recognin 3) E3
ubiquitin ligase regulates apoptosis by controlling the activity of
DIAP1 in Drosophila. UBR3 regulates cellular levels of
APE1/Ref1, a protein essential for DNA damage repair and transcription,
thus indirectly affect DNA damage repair pathway. Knockout
of Ubr3 in MEF led to genomic instability and cell death (
Recent report identified Ubr3 as a novel, positive regulator of
Hedgehog (Hh) signaling in Drosophila and vertebrates through
Ubiquitylation of Costal2/Kif7, a central component of Hh
). As haploinsufficiency of Patched-1, a gene that encodes
a repressor of the Hh pathway, dysregulates the Hh pathway
and increases genomic instability, it was suggested that
inappropriate Hh pathway activation may promote tumorigenesis by
disabling a key signaling pathway that helps maintain genomic
stability and inhibits tumorigenesis (
Mutated genes for which little information is available
Limited information is available for the functions of IGSF10 and
ZNF226. IGSF10 (immunoglobulin superfamily member 10) and
ZNF226 (zinc finger protein 226) are involved in loss of function
and both have a mutation rate of 5% in HCC.
After examining the functions of the frequently mutated genes
from a genomic instability standpoint, a picture emerged. First,
HCC development is facilitated by the activation of oncogenic
pathways, such as the Wnt (beta-catenin, Axin1 and ZIC3), Smad
(WWP1) and EGFR (MIG6) pathways. Further, maintaining both
the oxidative stress response (i.e. NFE2L2) and efficient DNA
damage checkpoint and repair (i.e. TP53, ATM, RPS6KA3, ARID1A and
ARID1B) pathways is important to prevent the genomic
instability that leads to HCC. Combined defects in the oxidative stress
response and DNA damage checkpoint and repair pathways in
particular result in high DNA damage and genomic instability.
Thus, we suggest a few approaches that may be administered
individually or simultaneously: (i) to attenuate Wnt and/or EGFR
signaling, (ii) to reduce ROS through antioxidants
supplementation and (iii) to reduce DNA damage by controlling exposure to
DNA-damaging agents or other DNA damaging events, such as
inflammation or alcohol consumption. Alternatively, biomarker
identification and immunotargeting or small molecule-mediated
approaches may be used to eliminate cells with defects in these
pathways. The pathway analysis should be refined and risk
factors should be considered to optimize prevention approaches.
This work was supported by grants from the U.S. NIH NCI
(no. R01CA094962 to C.V.R), the U.S. V.A. merit (grant no.
1I01BX003198-01 to C.V.R), and the Stephenson Cancer Center
We thank Kathy Kyler for editorial help.
Conflict of Interest Statement: None declared.
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