Cancer cell reprogramming to identify the genes competent for generating liver cancer stem cells
Wuputra et al. Inflammation and Regeneration
Cancer cell reprogramming to identify the genes competent for generating liver cancer stem cells
Kenly Wuputra 1
Chang-Shen Lin 1
Ming-Ho Tsai 1
Chia-Chen Ku 1
Wen-Hsin Lin 1
Ya-Han Yang 0
Kung-Kai Kuo 0
Kazunari K. Yokoyama 0 1
0 Center of Stem Cell Research, Kaohsiung Medical University , Kaohsiung 807 , Taiwan
1 Graduate Institute of Medicine, Kaohsiung Medical University , Kaohsiung 807 , Taiwan
The cancer stem cell (CSC) hypothesis postulates that cancer originates from the malignant transformation of stem/ progenitor cells and is considered to apply to many cancers, including liver cancer. Identification that CSCs are responsible for drug resistance, metastasis, and secondary tumor appearance suggests that these populations are novel obligatory targets for the treatment of cancer. Here, we describe our new method for identifying potential CSC candidates. The reprogramming of cancer cells via induced pluripotent stem cell (iPSC) technology is a novel therapy for the treatment and for the study of CSC-related genes. This technology has advantages for studying the interactions between CSC-related genes and the cancer niche microenvironment. This technology may also provide a useful platform for studying the genes involved in the generation of CSCs before and after reprogramming, and for elucidating the mechanisms underlying cancer initiation and progression. The present review summarizes the current understanding of transcription factors involved in the generation of liver CSCs from liver cancer cell-derived iPSCs and how these contribute to oncogenesis, and discusses the modeling of liver cancer development.
c-JUN oncogene; Induced pluripotent stem cells; Liver cancer; OCT4; Reprogramming
The cancer stem cell (CSC) hypothesis initially proposed
for leukemia by J. Dick is now accepted [1–3]. CSCs
represent a small subset of cells within a tumor that are
endowed with stem-like properties such as the ability for
(i) self-renewal, (ii) pluripotency, (iii) tumor formation,
and (iv) drug resistance [4, 5]. Importantly, CSCs are
thought to be responsible for tumor initiation,
recurrence, and metastasis through the reduced sensitivity of
cancer cells to chemotherapy compared with that of the
original tumor cells . Growing evidence about liver
CSCs confirmed their resistance to therapeutic drugs
such as cisplatin, 5-fuluorouracil, and so on .
The primary strategy for inducing liver CSCs is to first
enrich the cells using classical stem cell markers of stem
cells such as CD13, CD24, CD44, CD47, CD90, CD133,
epithelial cell adhesion molecule, and OV6, and then to
apply functional methodologies such as side-population
analysis, the ALDEFLUORTM assay, the sphere
formation, and so on [8–11]. This cell population is then
transplanted into immuno-deficient mice to examine its
in vivo tumorigenic potential [7–9], and the cells are
studied further according to their expression of various
genes or signals such as Wnt, Notch, Hedgehog,
Transforming growth factor β, epithelial mesenchymal
transition (EMT)/mesenchymal epithelial transition
(MET) signaling, epigenetic regulators, and microRNAs.
The putative CSC subpopulation capable to initiating
tumor development at lower cell numbers are tested
further for self-renewal capacity using serial dilution of cells
to identify the CSCs. Cancers are also generated as a
consequence of transformation of the “driver” mutation
at initiation [12–15], and then positive selection and
clonal progression lead to the accumulation of
“passenger” mutations required for additional growth
advantages. It is generally accepted that a novel strategy
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is needed for the functional evaluation of both putative
driver and passenger mutations, and for studying their
molecular dynamics underlying cancer development.
Current cancer cell-reprogramming techniques such as
somatic cell nuclear transfer  and the generation of
induced pluripotent stem cells (iPSCs) [17–19] are used to
identify oncogenic genes. In 2006, Yamanaka et al. 
reported on the reprogramming of somatic cell into
induced pluripotent stem cell-like cells (iPSLCs). The
success in reprogramming a somatic cell into a stem cell-like
state has led to the idea of reprogramming malignant cells
back to their original state, which is well before the
oncogenic transformation occurs. The generation of iPSCs
from cancer cells may provide tools for exploring the
mechanisms of tumor initiation and progression in vitro,
for studying the heterogeneity and origin of CSCs, and for
producing cancer type-specific drug discovery. However,
these reprogramming methods remain a challenge
because of the cancer-specific epigenetic state and
chromosomal aberrations of cancer cells.
The epigenetic memory of the original cell type is
important to reprogramming and is closely related to
the inefficient reprogramming caused by failure of reset
the epigenome to an embryonic stem cell (ESC)-like
state . The epigenetic state is reversible, but attempts
to reprogram cancer cells have produced incomplete
resetting of the cancer-associated epigenome because of
tumor heterogeneity and further accumulation of
oncogenic mutations. Therefore, cancer cell reprogramming
is currently limited to certain cancer types and
cancerspecific marks in the epigenome, which impede
successful reprogramming, and the underlying mechanism has
not been fully elucidated. The technique of cancer cell
reprograming is one possible approach for identifying
the committed genes of CSCs and for studying the
mechanisms underlying the transcriptional, translational,
and epigenetic inheritance of cancer development. The
characterization of epigenome and tumor suppressor
gene complexes, such as p53, p21Cip1, p27Kip1, the Ink4
family, and the polycomb repressive complex (PRC)
might identify the possible candidates for genes for
reprogramming of cancer cells to CSCs.
Obstacles to cancer cell reprogramming
The reprogramming of cancer cells is less successful
than the reprogramming of somatic cells [21–23].
Despite the presence of genetic alterations, melanoma
cells can be reversed to the pluripotent state, such as
that of the ESC, by nuclear transfer  (Table 1).
However, the cells involved in other malignant cancer
types, including breast cancer, leukemia, and
lymphoma, cannot be reprogrammed, which suggests
the presence of unknown barriers to reprogramming
[23, 25, 26]. Similarly, embryonic carcinoma (EC) cell
lines can be reprogrammed by nuclear transfer and
exhibit normal preimplantation development.
However, abnormal phenotypes of the clones occur,
which suggests that EC cell lines possess a unique set
of modifications in the epigenetic state that cannot be
reprogrammed by altering the developmental potential
. The limited number of studies that have
reported successful derivation of iPSCs from cancer
cells supports the notion that the existence of
cancerspecific genetic and epigenetic states hinders
successful reprogramming independently of the complexity of
the techniques used for reprogramming.
Reprograming studies using patient-derived cancer cells have
produced solid evidence of the capacity for reversal of
the malignant phenotype, and this method holds great
promise for breaking the seemingly irreversible state
associated with cancer.
Table 1 Summary of studies of reprogramming of cancer cells from induce pluripotent stem cell technology. We have modified the
summary of the table reported by Camare et al. [25, 26]
Utikal et al. (2009) 
Mice melanoma R454
Carette et al. (2010)  Human leukemia
Miyoshi et al. (2010)  Human gastrointestinal Retrovirus and lentivirus
cancer cells + lipofectamine OSKM
Kuo et al. (2016) 
Human HepG2 liver Lentivirus OSKM +
cancer cells and mouse shp53RNA (lentivirus O +
hepatocytes-iPSCs c-JUN for direct reprogramming)
Stemness characteristics and oncogenic functions of CSCs
Generation of iPSCs from cancer cells requires the
identification of genes that regulate stem cell self-renewal
and pluripotency such as OCT4, SOX2, and NANOG.
CSCs require oncogenes or tumor suppressor genes to
express oncogenic functions. Both expression states of
stemness genes and oncogenes/tumor suppressor genes
should be reorganized at the chromatin state by their
regulators of epigenetic modification. Thus, three classes
of gene sets might provide clues for understanding the
reprogramming of cancer cells.
OCT4 is known to be overexpressed in hematological
cancers, seminomas, and cancers of the bladder, brain,
lung, ovary, pancreas, prostate, kidney, and testicle .
OCT4 works synergistically with SOX2 via direct binding
to transactivate the target genes . Both SOX2 and
OCT4 are activators of genes involved in pluripotency,
including themselves and NANOG , and repressors of
genes involved in differentiation [31, 32]. Both SOX2 and
OCT4 regulate their own transcription by binding the
composite elements of SOX–OCT in their enhancers .
Overexpression of SOX2 is detected in recurrent
prostate cancer, head and neck squamous cell carcinoma,
glioblastoma, small-cell lung cancer, and cancers of the
breast, liver, pancreas, and stomach . Overexpression
of SOX2 increases cell proliferation via cyclin D3, and
represses cell cycle regulators such as p21Cip1 and
p27Kip1 . SOX2 promotes the invasion, migration,
and metastasis of melanoma, colorectal cancer, glioma,
and cancers of the stomach, ovary, and liver through the
activation of matric metalloproteinases family, and
phosphatidylinositol 3-kinase (PI3K)–RAC-α
serine/threonine kinases (AKT)–mammalian target of the rapamycin
signaling pathway [35–37].
NANOG is overexpressed in oral squamous cell
carcinoma and other types of cancers . NONOG is capable
of maintaining pluripotency of ESCs independently of the
leukemia inhibitory factor-signal transducers and activator
of transcription pathway, which is different from the case
of OCT4 [38, 39]. NANOG also controls the cell cycle
and proliferation by directly binding to the cyclin D1
promoter for transactivation . NANOG induced the
expression of cancer-related genes like CD133 and
aldehyde dehydrogenase 1A1 . These stemness
transcription factors of SOX2, OCT4, and NANOG co-occupy the
promoter regions of about 350 genes in the genome, and
OCT4 occupies more than 90% of the promoter regions
bound by the OCT4 and SOX2 in human ESCs. These
findings suggest that the OCT4–SOX2–NANOG axis is
the key cascade for stemness .
Reprograming of cancer cells using iPS technology
It has been proposed that oncogenes and tumor
suppressor genes should be activated or repressed to
generate CSCs. However, the actual oncogenes that can
generate CSCs have not been characterized.
Carette et al.  reprogrammed a cell line derived
from chronic myeloid leukemia (CML) by infecting them
with a retrovirus that induced the expression of OCT4,
SOX2, KLF4, and MYC (OSKM) followed by the
subcutaneous injection of the CML-iPSCs into nonobese/
diabetic severe combined immunodeficient (NOD-SCID)
mice [Table 1]. They found that the teratomas produced
contained differentiated cells in three germ layers, which
indicated pluripotency. Whereas the parental CML cell
lines were dependent on the BCR–ABL pathway, by
contrast, the CML iPSCs were independent of this
BCR–ABL signaling and showed resistance to imatinib.
However, Cratte et al. did not identify the signaling
pathway involved in the suppression of this BCR–ABL
cascade. Miyoshi et al.  reported on the
reprogramming of gastrointestinal cancer cell lines into iPSCs
through the OSKM method [Table 1]. Tumors were
generated by parenteral injection of gastrointestinal
cancer cells into NOD-SCID mice, but not by injection of
differentiated cells arising from the iPSCs. These iPSCs
expressed increased levels of tumor suppressor genes
such as p16Ink4a and p53 upon differentiation.
Striker et al.  reported the reprogramming of
glioblastoma (GBM) cells to neural stem cells (NSCs) by
PiggyBac transposon vectors that expressed OCT4 and
KLF4. In these GBM iPSCs, the widespread resetting of
epigenetic methylation occurred in cancer-specific
methylation variable positions, the GBM tumor
suppressor gene CDKN1C (p57Kip2), and testin LIM domain
protein (TES). The neural progenitor cells (NPCs)
differentiated from GBM iPSCs resembled aggressive GBM
cells when transplanted into the adult mouse brain .
By contrast, non-neural mesodermal progenitors from
GBM iPSCs with sustained expression of TES and
CDKN1C formed benign tumors, and failed to infiltrate
the surrounding regions. These findings suggest that
DNA methylation is critical to the expression of these
particular genes. Kim et al.  generated the iPSC-like
cells from pancreatic ductal adenocarcinoma (PDACs)
by introducing the genes encoded OSKM. One cancer
iPS-like clone harbored classical PDAC mutations,
including kRAS and p16Ink4a heterozygous deletions and
decreased SMAD copy number, and retained the
chromosomal alterations seen in the parental cells, and
differentiated into all three germ layers during in vitro
differentiation in the descendants, although the neural
lineages were underrepresented. An in vivo teratoma
showed that the iPSC-like cells generated multiple germ
layers tissues but preferred to generate endodermal
DNA methylation is another critical epigenetic change.
However, the reprogramming of cancer cells into iPSCs
shows that only about 50% of cancer-associated
epigenetic defects, as defined by a comparison between normal
and tumor cells, are stably reset, which suggests that
locus-specific changes in the epigenome are required.
Reconfiguration of the network of cell-fate transcription
factors and downstream developmental epigenetic
mechanisms, may effectively silence the cancer-promoting
pathways essential for uncontrolled proliferation and
infiltration. However, the reciprocal interaction of
epigenetic changes and driver mutations of cancer need to
be explored in greater detail.
Funato et al.  used the pluripotent stem cells to
study the role of driver mutations in modeling pediatric
brain tumors. Differentiation of human ESCs into NPCs
and induced cellular transformation through the
overexpression of a constitutively active PDGFRA, TP53
knockdown, or expressing the K27M mutant of histone
H3.3 found in pediatric gliomas. Moreover, induced
NPC transformation was induced by the combination of
different mutations typically observed in human gliomas
that collectively affect the signaling of the PI3K, MAPK,
and p53 pathways .
BMI1 of PRC1 has been shown to be involved in the
maintenance and/or self-renewal of many types of stem
cells, including embryonic, neural, hematopoietic, and
prostate stem cells . BMI1 promotes the proliferation
of leukemic stem cells in a mouse model , and
activates the self-renewal ability of NSCs . BMI1 is
known to be directly responsible for the regulation of
multiple targets such p16Ink4a and p14Arf  and to
bind directly to the promoter of PTEN gene, which
results in the activation of the PI3K–AKT signaling and
subsequent stabilization of SNAIL to induce the EMT
. BMI1 also occupies the cadherin promoter, which
causes E-cadherin repression  and cooperates with
TWIST1 to promote cancer dedifferentiation and
metastasis . In endometrial cancer cells, the loss of BMI1
results in the reduced expression of SOX2 and KLF4
. Overexpression of BMI1 correlates with
overexpression of NANOG, high tumor grade status, and
increased self-renewal in breast adenocarcinomas .
Recently, Kaufhold et al.  reported the
association of Yin Yang 1 (YY1) and CSC transcription
factors and that YY1 might be a transcriptional
repressor that acts on CSC-associated transcription
factors such as BMI1, SOX2, and OCT4. They also
proposed the existence a regulatory loop involving
crosstalk between the nuclear factor kB–PI3K–AKT
pathway and the downstream controls of target gene
products such as YY1, OCT4, SOX2 and BMI1. Taken
together, these findings suggest that most of the genes
critical for cancer reprogramming belong to the
families of (i) stemness genes, (ii) oncogenes/tumor
suppressor genes, and (iii) epigenetic-related genes of
DNA or histone modification, and (iv) the INK4 locus
mediated by PRC family.
Thus, we hypothesized a “two-hit” theory for the
generation of CSCs. The first hit introduces stemness and the
second hit induces oncogenic features or repression of
tumor suppressor function. Both are required for
induction and maintenance of CSCs by epigenetic alterations
induced by both genes. In this model, the reprogramming
can be used as a platform for identification of the
functional driver or passenger mutations, and modifications
under the activation of stemness genes and the activation
of oncogenes (or the repression of tumor suppressor
genes) (Fig. 1).
Selective plasticity of CSCs
Cell plasticity is a key issue for the reprogramming of
CSCs. Mu et al.  and Ku et al.  independently
reported the role of cell plasticity in cell identity, which
allows cancers to thrive. It has been known that
hormone deprivation therapy that suppresses androgen
receptor (AR) signaling is one of the treatments for
metastatic prostate cancer. However, prostate cancers
can become resistant to these drugs by losing hormone
dependence on androgen. Because androgen stimulates
Fig. 1 Schematic representation of hypothetical two-hit theory
for crosstalk to generate cancer stem cells by reprogramming.
Hypothetical two-hit theory for induction of cancer stem cells
was represented. Stemness factors (OCT4, SOX2, and NANOG)
and oncogene/tumor suppressor genes (oncogenes such as
Myc, KLF4, c-JUN, kRAS, etc.; antioncogenes such as p53, Rb,
PTEN, BMI1, EZH2, INK4 family, etc.) and epigenetic modification
of DNA methylation and histone modification are required for
generation of cancer stem cells by reprogramming. We have
reported the feedback control of c-JUN and OCT4 is critical for
generation of cancer stem like cells . The oncogene c-JUN
transactivated genes encoding OCT4, SOX2, and NANOG , and the
genes of OCT4, SOX2, and NANOG formed the molecular circuitry for
stemness and pluripotency , and then OCT4 upregulated the
expression of c-JUN gene to form the feedback circuit . Taken
together, we hypothesize that these feedback circuit might be
regulated by the family of the stemness genes and the family of
cancer-related oncogenes or tumor suppressor genes
the growth of prostate cancer cells, decreased
production of androgen and/or inhibition of the hormone’s
action on prostate cancer cells can make a tumor shrink
or grow more slowly. Mu et al.  reported that the
induction of SOX2 expression subsequent to loss of RB1
and TP53 contributes to neuroendocrine differentiation
and androgen independence of prostate cancer cells. Ku
et al.  examined the effects of RB1 and TP53
deletion in a mouse model of metastatic prostate cancer
(PTEN loss) and found that EZH2 inhibition restored
enzalutamide responsiveness in RB1- and TP53-depleted
and AR expressing LNCaP cells. The cell lineage
plasticity was determined by the E2F-regulating genes such as
SOX2 and EZH2. These data suggest that both tumor
suppressor genes, such as RB1, TP53, and PTEN, and
the cell cycle regulator E2F-controlled genes, such as
SOX2 and EZH2, might be critical for determination of
cell lineage plasticity.
Clone selection of CSCs from iPSC-like cells
In an attempt to isolate the CSCs, we have used a new
strategy to isolate the clone responsible for generating
CSCs among the heterogeneous clones of iPSC-like cells
derived from human hepatocyte cell lines . Using the
original four Yamanaka’s factors plus small hairpin TP53
plasmid, we have generated iPSC-like clones. To identify
the possible CSC clones, we have reduced the number of
cells that remain competent after tumor formation, as
determined by the transplantation of colonies of
xenograft-derived iPSC-like cells. One colony comprising
about 200 cells finally generated tumors in more than 40%
of transplants. We then characterized this colony to
determine its tumor-forming capacity. This clone exhibited
greater tumor-forming activity and other cancer-related
activities such as sphere formation, colony formation,
invasion activity, and drug resistance compared with the
original liver cancer cells.
To gain further insight into the acquired CSC
characteristics of this CSC colony, RNA sequencing was performed
and showed that OCT4 and c-JUN expression was greater
in this clone than in the original cancer cells. This
experiment suggests that both OCT4 and c-JUN are key factors
required for the feedback control of each other. Moreover,
the two genes—one a stemness gene and the other an
oncogene—were also competent in inducing CSCs derived
from iPSCs from mouse hepatocytes infected with
lentivirus that encoded OCT4 and c-JUN. This combination is
interesting because c-JUN is an oncogene and OCT4 is a
stemness gene. By themselves, OCT4 and c-JUN showed
less transformation activity, but the feedback between
OCT4 and c-JUN increased the likelihood of cancer
induction. Therefore, we hypothesize that both genes are
required to generate liver cancer and that the feedback
This strategy of cancer reprograming is one possible
approach for generating CSCs. Chang et al.  reported
that c-JUN is activated in pluripotent stemness gene
promoters such as OCT4, SOX2, and NANOG and
promotes the EMT in head and neck cancer cells. We have
introduced OCT4 and c-JUN into mouse iPSCs from
normal hepatocytes to generate tumor formation .
These approaches to induce the feedback regulation of
the stemness gene family and oncogene family might
provide a novel approach to generate CSC-like clones.
Elevated expression of OCT4 and c-JUN was observed
in specimens taken from patients with liver cancers.
These findings suggest that both genes are possible
candidates as future therapeutic targets.
In this review, we have described the methods for
generating CSC-like cells from iPSCs from liver cancer cells.
The critical point is to isolate the clone with strongest
tumor-inducing activity. We have identified the genes of
stemness and oncogenes as possible CSC target genes.
This approach can now be extended to isolate CSCs and
to generate disease- or cancer-specific models with
distinct features. However, the efficiency of cell
reprogramming from iPSCs to CSCs is lower and each CSCs
has been shown to be heterogeneous. Moreover, oncogene
induced plasticity including the CSC markers, and the
microenvironments controlling this process have still not
been elucidated, especially in the cases of the solid tumors
[23, 61–63]. Further work is needed to identify and
characterize CSC-like cells in other cancers and diseases,
which will help to detect the driver and passenger
mutations for generating cancers and genetic diseases.
The study of the reprogramming of cancer cells should be
encouraged to further progress the understanding of
cancer and disease biology.
CSC: Cancer stem cell; EC: Embryonic carcinoma; EMT: Epithelial
mesenchymal transition; ESC: Embryonic stem cell; GMB: Glioblastoma;
iPSC: Induced pluripotent stem cell; MET: Mesenchymal epithelial transition;
MMP: Matrix metalloproteinase; NOD SCID: Nonobese diabetic/severe
combined immunodeficient; OSKM: OCT4, SOX2, KLF4 and MYC;
PRC: Polycomb repressive complex; YY1: Yin Yang 1
This work was supported in part by MOST 104-2320-B-037-033-My2, and
MOST 104-2314-B-033-002 from the Ministry of Science and Technology;
NHRI-Ex102-10109B1, and NHRI-Ex104-10416S1, from the National Health
Research Institutes in Taiwan; and KMU-TP103G00, G01, G03, G04, G05,
KMU-TP103A104, KMU-TP104A04, KMU-TP104E24, KMU-TP104PR22, and
KMU-DT104001 from Kaohsiung Medical University in Taiwan.
KW and CSL contributed to the preparation of this review. All authors KW,
CSL, MHT, YHY, CCK, WHL, and KKK read and approved the final manuscript.
KKY prepared and revised the final manuscript.
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