Reprogramming of gastrointestinal cancer cells
DyahLaksmi Dewi,1,2,4Hideshi Ishii,1,4Naotsugu Haraguchi,1,4Shimpei Nishikawa,1,2Yoshihiro Kano,1,2
Takahito Fukusumi,1,2Katsuya Ohta,1,2Susumu Miyazaki,2Miyuki Ozaki,1,2Daisuke Sakai,1Taroh Satoh,1
Hiroaki Nagano,2Yuichiro Doki2and Masaki Mori2,3
Departments of1Frontier Science for Cancer and Chemotherapy,2Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan
(Received October 13, 2011 ⁄ Revised November 30, 2011 ⁄ Accepted December 5, 2011 ⁄ Accepted manuscript online December 12, 2011 ⁄ Article first published online January 17, 2012)
Cell reprogramming reverts cells to multipotent, preprogrammed
states by re-establishing epigenetic markers. It can also induce
considerable malignant phenotype modification. Because key
events in cancer relapse and metastasis, including epithelial–mes-
enchymal transition phenotypes, are regulated primarily by revers-
ible and transient epigenetic modifications rather than the
accumulation of irreversible and stable genetic abnormalities,
studying dynamic mechanisms regulating these biological pro-
cesses is important. Transcription factors for induced pluripotent
stem cells and non-coding microRNAs allow pluripotent phenotype
induction. We present the current knowledge of the possible
applications of cell reprogramming in reducing aggressive pheno-
type expression, which can induce tumor cell hibernation and
maintain appropriate phenotypes, thereby minimizing relapse and
metastasis after surgical resection of gastrointestinal cancer.
(Cancer Sci 2012; 103: 393–399)
acquire multidifferentiated characteristics following the loss and
re-establishment of important epigenetic markers including
DNA methylation.(1)This deregulation of important genomic
and epigenomic factors is commonly associated with the abnor-
mal cell differentiation characteristics of different cancers.(2–7)
Emerging data suggest that epigenetic modifications and cell
reprogramming-like processes are important for cellular trans-
formation and the development of malignant cancer pheno-
types.(8–12)Understanding the underlying process of epigenome
reprogramming facilitates the use of regenerative medicine and
cancer therapy. Here we discuss whether the reprogramming-
like phenomenon observed in normal cells can be adapted for
developing novel therapies.
uring cell reprogramming, mature cells revert to an imma-
ture, preprogrammed (undifferentiated) state, and usually
Programming and Reprogramming of Cells
Mammalian tissues develop from a totipotent zygote. During
cell differentiation, a less specialized cell (i.e. stem or progenitor
cell) continuously produces more specialized cell types through
cell division, and thus, a complex tissue system containing
increasingly differentiated and specialized cells is established.
Subsequently, pluripotent primitive ectodermal cells in the inner
cell mass of blastocysts develop from the totipotent zygote.(13,14)
Following blastocyst implantation, pluripotent epiblast cells dif-
ferentiate into somatic cells. Repression of the somatic program
and re-expression of pluripotency-specific genes through epige-
netic modifications are necessary for germ cell develop-
ment,(13,15)indicating that differentiated cells retain all the
genetic information necessary to generate an entire organism.
This was initially tested by cloning animals from differentiated
cells by nuclear transfer. Mouse(16,17)and human ES cells(18)are
derived from their respective blastocysts. The recently devel-
oped iPSCs(19,20)can produce derivatives of each germ layer.
Differentiation occurs both during the developmental stages and
in adults;(21)multipotent tissue stem cells produce completely
differentiated daughter cells during normal cell turnover in adult
tissues and during tissue repair.(22)Differentiation is associated
with dynamic alterations in cell morphology, cell metabolism,
and responsiveness to cell signaling,(21,23)which occur largely
because of highly regulated gene expression through mRNA
regulation(23)and non-coding miRNA expression.(24–26)
Definedfactor-mediated reprogramming. Considering the
ethical issues regarding the use of fertilized oocytes for estab-
lishing and producing ES cells, and the immunological compati-
bility that occurs in case of unrelated donors, a great
breakthrough was reported by Takahashi and Yamanaka(19),
who discovered that complete reprogramming can be achieved
by introducing defined biological factors, such as Oct4 (also
known as Pou5f1), Sox2, Klf4, and c-Myc, in mouse and human
fibroblasts.(20)The initial experimental injection of Fbx15-
selected iPSCs into mouse blastocysts revealed that iPSCs alone
could not efficiently produce chimeric mice, presumably
because of substantial methylation of immature gene (including
Nanog and Oct4) promoters.(19)Subsequent studies indicated
that modified selection methods of completely reprogrammed
cells through expression of endogenous Nanog(27,28)
Oct4(28)allowed the successful generation of viable chimeras
and detectable transmission into the germline.
Complete pluripotency. During stable Oct4 and Nanog selec-
tion, although the overall appearance of the colonies was simi-
lar,(27,28)quantitative differences existed between the two
selection strategies. Oct4-selected ES-like colonies provided
more stable and homogenous iPSC lines than Nanog-selected
ES-like colonies.(28)Eventually, the fraction of ES-like colonies
from Oct4-selected MEF cultures was two or threefold higher
than that from Nanog-selected cultures, although initially fewer
colonies existed with Oct4-selected MEF-derived iPSCs. This
suggests that although the Nanog locus was more easily acti-
vated, a higher fraction of colonies from Oct4-selected MEF
cells was reprogrammed to pluripotency.(28)These studies estab-
lished that selection for Oct4 and Nanog expression results in
germline-competent iPSCs with increased ES cell-like gene
expression and DNA methylation patterns compared with
Fbx15-iPSCs. Whereas one clone from seven Nanog-iPSC
clones was transmitted through the germline to the next genera-
tion,(27)Oct4-iPSCs injected into tetraploid blastocysts can gen-
erate live late-term embryos.(28)The biological potency and
epigenetic state of iPSCs and ES cells are the same. The overall
estimated efficiency (0.05–0.10%) to establish iPSC lines from
3To whom correspondence should be addressed.
4These authors contributed equally to this work.
ª ª 2011 Japanese Cancer Association
| vol. 103|no. 3|
MEFs was similar between Oct4 and Nanog selection, despite
the larger number of total Nanog-iPSC colonies(28)(Table 1).
Reprogramming barriers by tumor suppressors. Several fac-
tors can enhance the efficiency of iPSC generation, such as cell
cycle checkpoints mediated by the cyclin-dependent kinase
inhibitor family. The CDKN2b–CDKN2a locus on human chro-
mosome 9p21 (mice chromosome 4) is frequently lost in cancer.
The locus encodes three cyclin-dependent kinase inhibitors of
the cell cycle: p15INK4b, p16INK4a, and p14ARF (p19Arf in
mice) encoded by CDKN2b, CDKN2a, and an alternative read-
ing frame of CDKN2a, respectively.(29)These inhibitors are
endogenously expressed in differentiated cells and downregulated
Table 1. Summary of current studies of normal somatic cell reprogramming
Method for factor deliveryFactor Starting material Efficiency Ref.
Mouse embryonic and adult fibroblast
Human fetal fibroblast
10 colonies⁄5 · 104
198 colonies⁄0.9 · 106
Lower than the viral
OSKM MEF and hepatocyte(72)
OSKM Adult mouse liver and
Mouse neural stem cell
Slower than OK
100 · higher than
the OSKM method
20–50 · higher than the
direct infection method
OSKM Secondary somatic cells containing
Dox-inducible OSKM expression
(MEF, intestinal epithelium)
Adult human adipose stem cells
OSKM + VPA
OSK + VPA
OK + BIX⁄BayK
Slower kinetic than the viral
12 colonies⁄3.5 · 104
Neural progenitor cells
delivered by PiggyBac
Retrovirus for OSK
Transfection of miRNA
introduced by nucleofection
Murine and human embryonic
OSKM Human terminally differentiated
circulating T cells
Neonatal human epidermal
keratinocytes, HUVECs, and
amniotic fluid-derived cells
Primary human neonatal epidermal
keratinocytes, BJ human neonatal
foreskin fibroblasts, human fetal
lung fibroblasts, and human
fetal skin fibroblasts
Lentivirus Oct4 + small compound
4–6 colonies⁄1 · 106
Repeated transfection of
1.4%, 36-fold higher
miR-367 + VPA
Faster kinetics efficiency 2 ·
higher compared with OSKM
Efficiency 10000 · higher
Repeated transfectionHuman and mouse adipose
stromal cells, dermal fibroblast
5 colonies⁄5 · 104
of miRNA mimics
Addition of miRNA enhanced
efficiency by 4–6-fold
(miR-106b, 93), 3–4-fold
Addition of miRNA enhanced
efficiency by promoting MET
of miRNA mimics
BIX, the small molecule BIX-01294, an inhibitor of the G9a histone methyltransferase; K, Klf4; L, Lin28; M, c-Myc; MEF, mouse embryonic
fibroblast; MET, mesenchymal–epithelial transition; miR, microRNA; N, Nanog; n.d.: not determined; O, Oct3⁄4; Ref., reference; S, Sox2;
VPA, valproic acid, a HDAC inhibitor.
ª ª 2011 Japanese Cancer Association
by aberrant mitogenic signaling. The study of double KO
(Ink4ab) ⁄ )) and triple KO of all three ORFs (Cdkn2ab) ⁄ ))
showed that p15Ink4bcan act as a critical backup for p16Ink4a,
suggesting a rationale for frequent loss of the complete
CDKN2b–CDKN2a locus in human tumors.(30)Endogenous
p19Arf,(31)p16Ink4a,(32)and Trp53 (also known as p53), all inacti-
vated in several tumors,(33–35)can limit reprogramming and
inhibit pathways leading to an increased level of iPSC genera-
tion. In mice, Arf, rather than Ink4a, blocks important repro-
gramming pathways through p53 and p21 (encoded by Cdkn1a)
activation. However, in humans, INK4a is more important than
ARF.(32)Loss of replicative potential may prevent cell repro-
gramming. The acquisition of cell immortality is a rate-limiting
step for establishing pluripotency in somatic cells.(31,32)The
transient inhibition of these proteins may significantly improve
iPSC generation,(31–35)although the ability of the resultant iPS-
Cs to become tumorigenic is not completely understood. During
reprogramming, cells increase their intolerance to different types
of DNA damage. A p53-mediated DNA damage response limits
reprogramming to ensure iPSC genomic integrity and prevent
genomic instability.(35)This phenomenon emphasizes the simi-
larities between induced pluripotency and tumorigenesis. Even-
tually, approximately 20% of the offspring developed tumors
attributable to c-Myc transgene reactivation. Retroviral c-Myc
introduction should be avoided for clinical application.(27)Stud-
ies of the other barriers indicated that increased iPSC generation
efficiency is observed after treating cells with butyrate(36)or
vitamin C(37)or after exposing them to hypoxia.(38)
Reprogramming using miRNA. Considering the future appli-
cation of reprogramming technology, two major non-mutually
exclusive issues that should be solved are safety and efficiency.
The introduction and addition of specific non-coding miRNA(25)
can, for example, improve reprogramming efficiency.(9,39,40)
Regarding safety, genomic modification, which is critical to car-
cinogenesis, is an important concern. The introduction of genes
involved in reprogramming events is often facilitated by viral
vector-mediated transduction, which can involve random inser-
tions of exogenous sequences into the genome.(19,20)iPSCs can
be obtained using virus-free, removable PiggyBac transposons
or episomal systems,(41–44)but these approaches still use DNA
constructs; thus, the possibility of genomic integration of intro-
duced sequences is still a potential problem. Alternatively, the
Sendai virus has been used; iPSCs were generated from human
terminally differentiated circulating T cells(45)using Sendai
RNA virus vectors.(46,47)Reprogramming events using just pro-
tein or mRNA has also been reported, but the protocols involved
are technically challenging.(48–50)
Recently, two independent studies from the Morrisey group and
our group have demonstrated that human and mouse somatic cells
can bereprogrammed toiPSCs through forcedmiRNA expression,
completely eliminating the need for ectopic protein expres-
sion.(39,51)Morrisey group(39)revealed that lentiviral-mediated
transfection of immature miR-302⁄367 sequences generated
reprogrammed cells (miR-302⁄367 iPSCs) displaying characteris-
tics similar to those of Oct4⁄Sox2⁄Klf4⁄Myc-iPSCs, including
pluripotency marker expression, teratoma formation, and chimera
contribution and germline contribution for mouse cells. miR-367
expressionisrequired for miR-302⁄367-mediatedreprogramming,
activation of Oct4 expression, and Hdac2 suppression.(39)
Conversely, direct transfection of direct mature double-stranded
miRNAs (miR-200c + miR-302s + miR-369s) resulted in PSC
generation from differentiated adipose-derived stem cells in
humans and mice.(51)This reprogramming method does not
require vector-based gene transfer, and thus holds significant
potential in biomedical research and regenerative medicine.
Other reports have indicated that electroporation of the poly-
cistronic cassette of hsa-miR-302a⁄b⁄c⁄d resulted in the repro-
gramming of human hair follicle cells.(40)This reprogramming
mechanism functioned through miR-302-targeted cosuppression
of four epigenetic regulators: AOF2 (also known as KDM1 or
LSD1), AOF1, MECP1-p66, and MECP2.(40)Furthermore, ret-
roviral expression of the polycistronic cassette of hsa-miR-
302a⁄b⁄c⁄d allowed the development of iPSC-like phenotypes
from human skin cancer cells.(9)Because these methods were
carried out without transcription factors, the introduction of
miRNAs may play critical roles in differentiated cell reprogram-
ming in humans and mice.
The underlying mechanism of miRNA reprogramming is not
completely understood. Generally, miRNAs are involved in
translation inhibition, mRNA destabilization, and coding mRNA
function suppression.(52,53)We hypothesize that miRNA expres-
sion fine-tunes cell reprogramming mainly by inhibiting mRNA
signaling, although evidence also suggests that miRNAs may
have other functions including translation stimulation through
an unknown mechanism. For example, miR-369-3p, which was
used for reprogramming,(51)acts as a unique switch for regulat-
ing translation repression and activation.(54)miR-302,(39,40,51,55)
which targets TGFb receptor 2 and antagonizes EMT,(55)was
also reported to suppress AOF2, AOF1, MECP1-p66, and
MECP2,(40)indicating that the miR-302 pathway is fundamental
for reprogramming. Inhibition or reversion of EMT could be
stimulated by miR-302,(39,40,55)
200c.(51)TGFb modulates reprogramming by EMT signaling,
whereas Klf4 stimulated E-cadherin expression, a hallmark of
MET, which is involved in the stimulation of important repro-
gramming events.(56)When mammary epithelial cells, which
express endogenous Klf4 (MET expression is unnecessary),
were used as the starting material, iPSCs were successfully
developed only by introducing Sox2 and Oct4 without adding
Klf4.(56)This suggests that the requirements needed for EMT
inhibition may be dependent on cellular context.
Effect of reprogramming on cancer cells. Retrovirus-mediated
gene transfer in gastrointestinal cancer cells resulted in the
induction of ES-like gene and protein expression (patterns
induced from the endoderm of the gastrointestinal tract to the
mesoderm and ectoderm).(10)Interestingly, retrovirus-mediated
exogenous expression of Oct4⁄Sox2⁄Klf4⁄Myc or Oct4⁄
Sox2⁄Klf4 sensitized gastrointestinal cancer cells to vitamins
and other chemotherapeutic agents.(10)In vivo experiments
involving short-term cultured reprogrammed cells showed an
inhibition of tumorigenicity in DLD-1 colorectal cancer cells.(10)
The study also revealed changes in DNA methylation and his-
tone modification and revealed that the epigenome of DLD-1
cells resembled that of ES cells. The promoter region of
p16Ink4a was demethylated similar to the heavily demethylated
state.(10)Long-term cultured reprogrammed cells with gain-of-
function mutations, including TP53R175Hand KRASG12D, elicit a
malignant transformation with c-Myc activation in KRAS and
TP53-mutated HuCC-T1 cholangiocellular carcinoma cells, sug-
gesting a role of such oncogenic mutations in malignant pheno-
decreasing the p53 expression level enables the development of
murine fibroblasts into iPSCs capable of generating germline-
transmitting chimeric mice, indicating that p53 may not be nec-
essary for reprogramming. Silencing p53 will significantly
increase the reprogramming efficiency of human somatic
enhance defined factor-mediated cell reprogramming,(59)sug-
gesting that the TP53 mutation context is influenced by the qual-
ity and quantity of reprogramming events. Reprogramming
efficiency was increased in hypoxia,(38)an effect observed in
cancer cells (Masaki Mori, unpublished data, 2011).
Transfection of miR-302 induces ES-like phenotypes of skin
cancer.(9)MiR-302 also inhibits tumorigenecity by coordinating
suppression of the Cdk2 and Cdk4⁄6 cell cycle pathways.(60)
The study indicated that concurrent silencing of BMI-1, an
Dewi et al. Cancer Sci|
ª ª 2011 Japanese Cancer Association
| vol. 103| no. 3|
miR-302-targeted CSC marker, further promoted tumor suppres-
sor functions of p16Ink4a and p14⁄p19Arf directed against
Cdk4⁄6-mediated cell proliferation. Also, miR-302 inhibits
human pluripotent stem cell tumorigenicity by enhancing the
multiple G1phase arrest pathways.(60)Another study of glioma
indicated that the miR-302–367 cluster drastically affects the
Table 2 Summary of current studies of cancer cell reprogramming
MethodType of cancerMalignant-related phenotype Characterization Ref.
Suppressed proliferation, restore
normal differentiation, normal
proliferation in cultured blastocyst
Cloned blastocyst can support
postimplantation development, as
the embryo appeared normal and
showed extensive differentiation,
although not viable after E8.5
NT ES cells could form teratoma and
generate chimera. Injection into
tetraploid blastocyst resulted in a
normal embryo viable until E9.5
RAS+⁄Ink4a⁄Arf) ⁄ )
NT ES-cell chimeric mice developed
various types of tumors with shorter
latency and higher penetrance
compared with the donor mouse
Dependent on donor ECs, one NT ES
cell chimera suffered from head and
neck EC and was inviable, and the
other resulting NT ES cells showed a
broad differentiation potential into
teratomas and broad contribution to
Nuclei from EC can direct
resulting in normal appearing
blastocyst, higher efficiency of
producing an ES cell line compared
with the differentiated cells,
although the degree of
differentiation depends on the cell
Reduced invasion, tumor growth,
Downregulated Nodal signaling
through Lefty activation
Reduced migration ability, reduced
expression of cell cycle-related genes
(CCND1, CCND2, CDK2), and DNA
methylation facilitator, MeCP2
MECP1-p66, and some melanoma
Chimeras were tumor-free at
5 months of age
(miR-302a, b, c, d)
prostate cancer cell
Expression of pluripotency markers
Nanog, Oct4, Sox2, SSEA3, SSEA4
Demethylation of Oct4
Melanoma (R545) Teratoma (+), chimera (+), ES cell
marker expression, demethylation of
Nanog and Oct4
Expression of pluripotency marker,
demethylation of Nanog, in vitro
differentiation into adipocyte,
epithelial, mesenchymal, and neural
lineage, teratoma ())
Differentiated iPC (post-iPC) showed
sensitivity to chemotherapy, reduced
invasion, and reduced
tumorigenicity, showed higher
expression of p16 and p53 as
compared to the parental cell
Completely resistant to imatinib, loss
of BCR-ABL-dependent signaling
KBM7 cells derived
from blast crisis stage
A549 lung cancer
ES cell marker expression (+),
demethylation of Oct4 and Nanog,
Increased tumorigenic properties
when transplanted into a NOD⁄SCID
mouse, more aggressive and
invasive, teratoma ())
Demethylation of Oct4 promoter
expressed endogenous Nanog and
Oct4 although lower than HES cell
ALP(+), teratoma ())
Reprogramming efficiency was
higher compared with normal
primary lung fibroblast
Oocyte extract Breast cancer (MCF7
Re-expression of tumor suppressor
genes RARB, CST6, CCND2, CDKN2A
through demethylation and
remodeling of histone marks to a
more euchromatic state
No changes in DNA methylation at
pluripotency gene promoters
Reduced colony formation
ALP, alkaline phosphatase (staining); EC, embryonal carcinoma; HES, human embryonic stem; iPC, induced pluripotent stem (iPS)-like cancer cells; K,
Klf4; L, Lin28; M, c-Myc; N, Nanog; n.d., not determined; NT ES, nuclear transfer-generated embryonic stem cells; O, Oct3⁄4; Ref., reference; S, Sox2.
ª ª 2011 Japanese Cancer Association
self-renewal and infiltration properties of glioma-initiating cells
through Cxcr4 repression and consequent disruption of the Shh-
Gli-Nanog network.(61)This indicates that the miR-302–367clus-
ter can efficiently trigger a cascade of inhibitory events leading to
the disruption of CSC-like and tumorigenic properties.(61)Taken
together, further study of novel reprogramming-based therapeutic
approaches that could prove beneficial for treatment of tumors
with p53 inactivation(33,34,58)and⁄or of CSCs, which can survive
in aregion of hypoxia,(38)iswarranted (Table 2; Fig. 1).
Defined Factor-Induced Reprogramming and CSCs
The differential mechanisms between cancer cells, which
undergo a mutated form of reprogramming, and naturally occur-
ring CSCs remain unclear.
Gastrointestinal cancer cells. Recently, it has been proposed
that two types of stem cells coexist in normal and cancer cells and
that these stem cells are transiently regulated by epigenetic con-
trols.(62–65)Emergingevidence indicates that quiescent and active
stem cell subpopulations that are in lower metabolic and prolifer-
ative states, respectively, may coexist in several tissues.(62)It has
been proposed that these stem cell populations have separate but
cooperative functional roles, and these adult stem cells are crucial
for physiological tissue renewal and regeneration after injury.(62)
Generally, astemcelldividesasymmetrically intoanewstemcell
(self-renewal) and a committed progenitor (differentiation).
Whereas the asymmetric architecture of the stem cell niche in
Drosophila and Caenorhabditis elegans is apparent, mammalian
adult stem cells are generally detected in a predominantly quies-
cent state.(63,64)Quiescent stem cells have been proposed to pro-
duce transit-amplifying cells in rapidly regenerating tissue, which
differentiate into mature cells and provide tissue architecture.
Considering that transit-amplifying cells have a short lifespan
and limited self-renewal capabilities, recent studies suggest that
stem cell populations that are long-lived yet constantly cycling
are involved in the maintenance of tissue homeostasis.(62)A new
model describes the coexistence of quiescent and active adult
stem cell subpopulations in bone marrow, intestinal epithelium,
and hair follicles.(62)In contrast to physiological tissues, serial
transplantation experiments indicated that liver CSCs are com-
posed of quiescent and active CSCs. This system plays a role in
the exertion of resistance against chemoradiotherapy. During the
study of CSCs, we identified CD13+CSCs as a subpopulation of
quiescent stem cells of the liver.(65)Our study indicated that
TGFb induced the development of a CD13+CSC population
(Masaki Mori, submitted). CD13+CSCs express immature genes
often connected with a lower differentiation state, an observation
(Masaki Mori, unpublished data, 2011). Considering TGFb
signaling counteracts the induction of cell reprogramming from
normal differentiated cells, the outcome of reprogramming-like
stimulation should beinvestigated.
levels of ES-like genes could be relevant to tumor cell malig-
nancy.(66)The concept that a small population is contained in
adult tissues may be relevant to CSCs in a tumor.(67)The involve-
ment of a very small embryonic⁄epiblast-like stem cell popula-
tion in carcinogenesis could support century-old concepts
involving embryonic rest- or germline-origin hypotheses of can-
cer development;(67)however, this working hypothesis requires
further direct experimental confirmation.(67)Further evidence
indicates that tissues contain a unique population of mesenchy-
mal stem cells or Muse cells,(68)and that Muse cells are a primary
source of iPSCs in human fibroblasts.(69)By using immunocyto-
chemistry to express Nanog, Oct3⁄4, and Sox2 and TRA-1–81 to
assess reprogramming efficiency, the authors showed that iPSC
CSCs. Endogenous expression
mechanism of phenotype reversal of parental cells through the modulation of epigenetic status into a more undifferentiated state. Defined
transcription factors (Yamanaka cocktail)-induced reprogramming is involved in the regulation of mesenchymal–epithelial transition (MET),
which is controlled by a group of microRNAs (miR) through ZEB1⁄ZEB2 and TGFbR2. Those miRNAs play a role in global demethylation through
AOF1⁄2 and MECP1⁄2. In contrast to normal cell reprogramming (upper panel), cancer cell reprogramming (lower panel) remains obscure. The
reverse of MET, epithelial–mesenchymal transition (EMT), results in a chemotherapy-resistant phenotype. Thus, reprogramming is supposed to
open the silent chromatin through DNA demethylation and activate histone codes, which would elicit re-expression of tumor suppressor genes,
pushing cancer cells into a more benign phenotype. Further investigation would provide insight into how much of the tumor phenotype could
be reversed through the contribution of reversible epigenetic and irreversible genetic changes in cancer. Reprogramming cancer cells might
become a promising method for reversing or attenuating malignancy for therapeutic purposes. iPCC, induced pluripotent stem cell-like cancer
cell; iPSC, induced pluripotent stem cell; TGF, transforming growth factor; TSG, tumor suppressor gene.
Cellular reprogramming in normal and cancer cells. Cellular reprogramming in normal and cancer cells can be viewed globally as a
Dewi et al.Cancer Sci|
ª ª 2011 Japanese Cancer Association
|vol. 103| no. 3|
lines were generated with an efficiency of 0.001% from naive
human skin fibroblasts, whereas Muse-iPSCs were formed with
an efficiency of 0.03%, indicating that Muse cells generate iPSCs
30-fold more efficiently than naive fibroblasts.(69)This type of
subpopulation study elicits a challenging notion that a subset of
pre-existing adult stem cells in adult human tissues (or fibro-
blasts), which are somewhat similar to iPSCs, selectively become
iPSCs, whereas the remaining cells make no contribution to iPSC
generation.(69)Nevertheless, at least two issues should be consid-
ered. First, the efficiency of iPSC generation in this study is much
lower than that reported in other studies (‡0.02%; Table 1).(20,70)
Although the susceptibility to each cell reprogramming may be
presumably based on pre-existing conditions of epigenetic and
excluded without adjusting the complete reprogramming technol-
ogy. Second, given that higher efficiencies of reprogramming
have been reported (up to approximately 10%, see Table 1) than
the pre-existing frequency of Muse cells in tissues, (1.1–1.3% of
human fibroblasts or bone marrow stromal cells formed Muse
cell-derived cell clusters in naive populations without long-term
trypsin incubation), cells other than Muse cells may generate iPS-
Cs. Taken together, it may be too early to conclude whether the
defined factor-induced reprogramming fits the elite model,(69)
rather than the stochastic model of iPSC generation.(70)To recon-
cile these issues, further investigation is necessary to improve the
reprogramming efficiency and understand the mechanism by
which cellular reprogramming functions, especially in subpopu-
lations of susceptible clones subjected to defined factor-induced
reprogramming. Considering that ES-like genes expressing CSCs
and unique populations including very small embryonic⁄epi-
blast-like stem cells and Muse cells could be essential in cancer
development, further research is necessary to determine the pres-
ence of these cell subpopulations in tumor tissues, relevancy to
epithelial cancerous cells, and susceptibility of reprogramming
events in these cell populations.
Tissue homeostasis is a carefully balanced process controlled by
epigenome regulation and efficient interplay between stem cells,
their progeny, and the microenvironment (e.g. recently reviewed
in intestinal stem cells(23)). Epigenome deregulation and malig-
nant stem cell formation lead to tumor cell development. Repro-
gramming technology or epigenome modification through
transfection of iPSC factors can lead to ES-like gene expression
patterns and considerable malignant phenotype modifica-
tion,(10,60)indicating that this technology could be used to create
novel therapeutic targets against CSCs by combining small non-
coding RNAs with efficient drug delivery systems.
This work was supported in part by: a grant-in-aid for scientific research
from the Ministry of Education, Culture, Sports, Science, and Technol-
ogy, Japan (H.I., N.H., H.N., Y.D., M.M.); the Ministry of Health,
Labour, and Welfare; the Princess Takamatsu Foundation, Japan (H.I.,
M.M.); the Takeda Foundation, Japan (H.I.); the Senshin Medical
Research Foundation (H.I.); Chugai Pharmaceutical Corporation, Japan
(H.I., N.H. D.S., T.S.); and Yakult Corporation, Japan (H.I., N.H. D.S.,
T.S.). Patents pending on PCT⁄JP2011⁄053457, PCT⁄JP2011⁄054287,
Muse (cells) multilineage-differentiating stress-enduring
TGF transforming growth factor
cancer stem cell
induced pluripotent stem cell
mouse embryonic fibroblast
1 Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian
development. Science 2001; 293: 1089–93.
2 Vogelstein B, Kinzler KW. The multistep nature of cancer. Trends Genet
1993; 9: 138–41.
3 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: 57–70.
4 Barker N, Ridgway RA, van Es JH et al. Crypt stem cells as the cells-of-
origin of intestinal cancer. Nature 2009; 457: 608–11.
5 Irons RD, Stillman WS. The process of leukemogenesis. Environ Health
Perspect 1996; 104 (Suppl 6): 1239–6.
6 Bonnet D, Dick JE. Human acute leukemia is organized as a hierarchy that
originates from a primitive hematopoietic cell. Nat Med 1997; 3: 730–37.
7 Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and
cancer stem cells. Nature 2001; 414: 105–11.
8 Kasemeier-Kulesa JC, Teddy JM, Postovit LM et al. Reprogramming
multipotent tumor cells with the embryonic neural crest microenvironment.
Dev Dyn 2008; 237: 2657–66.
9 Lin SL, Chang DC, Chang-Lin S et al. Mir-302 reprograms human skin
cancer cells into a pluripotent ES-cell-like state. RNA 2008; 14: 2115–24.
10 Miyoshi N, Ishii H, Nagai K et al. Defined factors induce reprogramming
of gastrointestinal cancer cells. Proc Natl Acad Sci USA 2010; 107: 40–
11 Wang J, Emadali A, Le Bescont A, Callanan M, Rousseaux S, Khochbin S.
Induced malignant genome reprogramming in somatic cells by testis-specific
factors. Biochim Biophys Acta 2011; 1809: 221–5.
12 Ben-David U, Benvenisty N. The tumorigenicity of human embryonic and
induced pluripotent stem cells. Nat Rev Cancer 2011; 11: 268–77.
13 Surani MA, Hayashi K, Hajkova P. Genetic and epigenetic regulators of
pluripotency. Cell 2007; 128: 747–62.
14 Rasmussen TP, Corry GN. Epigenetic pre-patterning and dynamics during
initial stages of mammalian preimplantation development. J Cell Physiol
2010; 225: 333–6.
15 Surani MA, Durcova-Hills G, Hajkova P, Hayashi K, Tee WW. Germ line,
stem cells, and epigenetic reprogramming. Cold Spring Harb Symp Quant Biol
2008; 73: 9–15.
16 Evans MJ, Kaufman MH. Establishment in culture of pluripotential cells from
mouse embryos. Nature 1981; 292: 154–6.
17 Martin GR. Isolation of a pluripotent cell line from early mouse embryos
cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl
Acad Sci USA 1981; 78: 7634–8.
18 Thomson JA, Itskovitz-Eldor J, Shapiro SS et al. Embryonic stem cell lines
derived from human blastocysts. Science 1998; 282: 1145–7.
19 Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse
embryonic and adult fibroblast cultures by defined factors. Cell 2006; 126:
20 Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem
cells from adult human fibroblasts by defined factors. Cell 2007; 131:
21 Beers MF, Morrisey EE. The three R’s of lung health and disease: repair,
remodeling, and regeneration. J Clin Invest 2011; 121: 2065–73.
22 Herdrich BJ, Lind RC, Liechty KW. Multipotent adult progenitor cells: their
role in wound healing and the treatment of dermal wounds. Cytotherapy 2008;
23 Medema JP, Vermeulen L. Microenvironmental regulation of stem cells in
intestinal homeostasis and cancer. Nature 2011; 474: 318–26.
24 Houbaviy HB, Murray MF, Sharp PA. Embryonic stem cell-specific
MicroRNAs. Dev Cell 2003; 5: 351–8.
25 Mattick JS, Makunin IV. Non-coding RNA. Hum Mol Genet 2006; 1: R17–29.
26 Leung AK, Sharp PA. MicroRNA functions in stress responses. Mol Cell
2010; 40: 205–15.
27 Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced
pluripotent stem cells. Nature 2007; 448: 313–7.
28 Wernig M, Meissner A, Foreman R et al. In vitro reprogramming of
fibroblasts into a pluripotent ES-cell-like state. Nature 2007; 448: 318–24.
29 Gil J, Peters G. Regulation of the INK4b–ARF–INK4a tumour suppressor
locus: all for one or one for all. Nature Rev Mol Cell Biol 2006; 7: 667–77.
30 Krimpenfort P, IJpenberg A, Song JY, van der Valk M, Nawijn M,
Zevenhoven J. Berns A. p15Ink4b is a critical tumour suppressor in the
absence of p16Ink4a. Nature 2007; 448: 943–6.
31 Utikal J, Polo JM, Stadtfeld M et al. Immortalization eliminates a roadblock
during cellular reprogramming into iPS cells. Nature 2009; 460: 1145–8.
ª ª 2011 Japanese Cancer Association
32 Li H, Collado M, Villasante A et al. The Ink4⁄Arf locus is a barrier for iPS Download full-text
cell reprogramming. Nature 2009; 460: 1136–9.
33 HongH, Takahashi K,Ichisaka
pluripotent stem cell generation by the p53-p21 pathway. Nature 2009;
34 Kawamura T, Suzuki J, Wang YV et al. Linking the p53 tumour suppressor
pathway to somatic cell reprogramming. Nature 2009; 460: 1140–4.
35 Mario ´n RM, Strati K, Li H et al. A p53-mediated DNA damage response
limits reprogramming to ensure iPS cell genomic integrity. Nature 2009; 460:
36 Mali P, Chou B.-K, Yen J et al. Butyrate Greatly Enhances Derivation of
Human Induced Pluripotent Stem Cells by Promoting Epigenetic Remodeling
and the Expression of Pluripotency-Associated Genes. Stem Cell 2010; 28:
37 Esteban MA, Wang T, Qin B et al. Vitamin C Enhances the Generation of
Mouse and Human Induced Pluripotent Stem Cells. Cell Stem Cell 2010; 6:
38 Yoshida Y, Takahashi K, Okita K, Ichisaka T, Yamanaka S. Hypoxia
enhances the generation of induced pluripotent stem cells. Cell Stem Cell
2009; 5: 237–41.
39 Anokye-Danso F, Trivedi CM, Juhr D et al. Highly efficient miRNA-
mediated reprogramming of mouse and human somatic cells to pluripotency.
Cell Stem Cell 2011; 8: 376–88.
40 Lin SL, Chang DC, Lin CH, Ying SY, Leu D, Wu DT. Regulation of somatic
cell reprogramming through inducible mir-302 expression. Nucleic Acids Res
2011; 39: 1054–65.
41 Okita K, Nakagawa M, Hyenjong H, Ichisaka T, Yamanaka S. Generation of
mouse induced pluripotent stem cells without viral vectors. Science 2008; 322:
42 Kaji K, Norrby K, Paca A, Mileikovsky M, Mohseni P, Woltjen K. Virus-free
induction of pluripotency and subsequent excision of reprogramming factors.
Nature 2009; 458: 771–5.
43 Woltjen K, Michael IP, Mohseni P et al. piggybac transposition reprograms
fibroblasts to induced pluripotent stem cells. Nature 2009; 458: 766–70.
44 Jia F, Wilson KD, Sun N et al. A nonviral minicircle vector for deriving
human iPS cells. Nat Methods 2010; 7: 197–9.
45 Seki T, Yuasa S, Oda M et al. Generation of induced pluripotent stem cells
from human terminally differentiated circulating T cells. Cell Stem Cell 2010;
46 Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of
transgene-free human pluripotent stem cells using a vector based on Sendai
virus, an RNA virus that does not integrate into the host genome. Proc Jpn
Acad Ser B Phys Biol Sci 2009; 85: 348–62.
47 Ban H, Nishishita N, Fusaki N et al. Efficient generation of transgene-free
human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai
virus vectors. Proc Natl Acad Sci USA 2011; 108: 14234–9.
48 Kim D, Kim CH, Moon JI et al. Generation of human induced pluripotent
stem cells by direct delivery of reprogramming proteins. Cell Stem Cell 2009;
49 Zhou H, Wu S, Joo JY et al. Generation of induced pluripotent stem cells
using recombinant proteins. Cell Stem Cell 2009; 4: 381–4.
50 Warren L, Manos PD, Ahfeldt T et al. Highly efficient reprogramming to
pluripotency and directed differentiation of human cells with synthetic
modified mRNA. Cell Stem Cell 2010; 7: 618–30.
51 Miyoshi N, Ishii H, Nagano H et al. Reprogramming of mouse and human
cells to pluripotency using mature microRNAs. Cell Stem Cell 2011; 8:
52 Ambros V. The functions of animal microRNAs. Nature 2004; 431: 350–5.
53 John B, Enright AJ, Aravin A, Tuschl T, Sander C, Marks DS. Human
MicroRNA targets. PLoS Biol 2004; 2: e363.
54 Vasudevan S, Tong Y, Steitz JA. Switching from Repression to Activation:
microRNAs Can Up-Regulate Translation. Science 2007; 318: 1931–4.
55 Liao B, Bao X, Liu L et al. MicroRNA cluster 302-367 enhances somatic cell
reprogramming by accelerating a mesenchymal-to-epithelial transition. J Biol
Chem 2011; 286: 17359–64.
56 Li R, Liang J, Ni S et al. A mesenchymal-to-epithelial transition initiates and
is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell
2010; 7: 51–63.
57 Nagai K-i, Ishii H, Miyoshi N et al. Long-term culture following ES-like
gene-induced reprogramming elicits an aggressive phenotype in mutated
cholangiocellular carcinoma cells. BiochemBiophysic Res Commun 2010; 395:
58 Zhao Y, Yin X, Qin H et al. Two supporting factors greatly improve the
efficiency of human iPSC generation. Cell Stem Cell 2008; 3: 475–9.
59 Moon JH, Ishii H, Dewi DL et al. Gain-of-function oncogenic mutations in
TP53 enhance defined factor-mediated cellular reprogramming. Nature
60 Lin SL, Chang DC, Ying SY, Leu D, Wu DT. MicroRNA miR-302 inhibits
the tumorigenecity of human pluripotent stem cells by coordinate suppression
of the CDK2 and CDK4⁄6 cell cycle pathways. Cancer Res 2010; 70: 9473–
61 Fareh M, Turchi L, Virolle V et al. The miR 302-367 cluster drastically
affects self-renewal and infiltration properties of glioma-initiating cells
through CXCR4 repression and consequent disruption of the SHH-GLI-
NANOG network. Cell Death Differ 2011; DOI: 10.1038/cdd.2011.89. [Epub
ahead of print].
62 Li L, Clevers H. Coexistence of quiescent and active adult stem cells in
mammals. Science 2010; 327: 542–5.
63 Arai F, Hirao A, Ohmura M et al. Tie2⁄angiopoietin-1 signaling regulates
hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004; 118:
64 Fuchs E, Segre JA. Stem cells: a new lease on life. Cell 2000; 100: 143–
65 Haraguchi N, Ishii H, Mimori K et al. CD13 is a therapeutic target in human
liver cancer stem cells. J Clin Invest 2010; 120: 3326–39.
66 Ben-Porath I, Thomson MW, Carey VJ et al. An embryonic stem cell-like
gene expression signature in poorly differentiated aggressive human tumors.
Nat Genet 2008; 40: 499–507.
67 Ratajczak MZ, Shin DM, Liu R et al. Epiblast⁄germ line hypothesis of cancer
development revisited: lesson from the presence of Oct-4+ cells in adult
tissues. Stem Cell Rev 2010; 6: 307–16.
68 Kuroda Y, Kitada M, Wakao S et al. Unique multipotent cells in adult
human mesenchymal cell populations. Proc Natl Acad Sci USA 2010; 107:
69 Wakao S, Kitada M, Kuroda Y et al. Multilineage-differentiating stress-
enduring (Muse) cells are a primary source of induced pluripotent stem cells
in human fibroblasts. Proc Natl Acad Sci USA 2011; 108: 9875–80.
70 Yamanaka S. Elite and stochastic models for induced pluripotent stem cell
generation. Nature 2009; 460: 49–52.
71 Yu J, Vodyanik MA, Smuga-Otto K et al. Induced Pluripotent Stem Cell
Lines Derived from Human Somatic Cells. Science 2007; 318: 1917–20.
72 Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced
pluripotent stem cells generated without viral integration. Science 2008; 322:
73 Aoi T, Yae K, Nakagawa M et al. Generation of pluripotent stem cells from
adult mouse liver and stomach cells. Science 2008; 321: 699–702.
74 Kim JB, Zaehres H, Wu G et al. Pluripotent stem cells induced from adult
neural stem cells by reprogramming with two factors. Nature 2008; 454: 646–
75 Huangfu D, Maehr R, Guo W et al. Induction of pluripotent stem cells by
defined factors is greatly improved by small-molecule compounds. Nat
Biotechnol 2008; 26: 795–7.
76 Wernig M, Lengner CJ, Hanna J et al. A drug-inducible transgenic system for
direct reprogramming of multiple somatic cell types. Nat Biotechnol 2008; 26:
77 Sun N, Panetta NJ, Gupta DM et al. Feeder-free derivation of induced
pluripotent stem cells from adult human adipose stem cells. Proc Natl Acad
Sci USA 2009; 106: 15720–5.
78 Shi Y, Do JT, Desponts C, Hahm HS, Scho ¨ler HR, Ding S. A combined
chemical and genetic approach for the generation of induced pluripotent stem
cells. Cell Stem Cell 2008; 2: 525–8.
79 Judson RL, Babiarz JE, Venere M, Blelloch R. Embryonic stem cell-specific
microRNAs promote induced pluripotency. Nat Biotechnol 2009; 27: 459–61.
80 Zhu S, Li W, Zhou H et al. Reprogramming of human primary somatic cells
by OCT4 and chemical compounds. Cell Stem Cell 2010; 7: 651–5.
81 Li Z, Yang CS, Nakashima K, Rana TM. Small RNA-mediated regulation of
iPS cell generation. EMBO J 2011; 30: 823–34.
82 Subramanyam D, Lamouille S, Judson RL et al. Multiple targets of miR-302
and miR-372 promote reprogramming of human fibroblasts to induced
pluripotent stem cells. Nat Biotechnol 2011; 29: 443–8.
83 Li L, Connelly MC, Wetmore C, Curran T, Morgan JI. Mouse embryos cloned
from brain tumors. Cancer Res 2003; 63: 2733–36.
84 Hochedlinger K, Blelloch R, Brennan C et al. Reprogramming of a melanoma
genome by nuclear transplantation. Genes Dev 2004; 18: 1875–85.
85 Blelloch RH, Hochedlinger K, Yamada Y et al. Nuclear cloning of embryonal
carcinoma cells. Proc Natl Acad Sci USA 2004; 101: 13985–90.
86 Postovit LM, Margaryan NV, Seftor EA et al. Human embryonic stem cell
microenvironment suppresses the tumorigenic phenotype of aggressive cancer
cells. Proc Natl Acad Sci USA 2008; 105: 4329–34.
87 Utikal J, Maherali N, Kulalert W, Hochedlinger K. Sox2 is dispensable for the
reprogramming of melanocytes and melanoma cells into induced pluripotent
stem cells. J Cell Sci 2009; 122: 3502–10.
88 Carette JE, Pruszak J, Varadarajan M, Blomen VA, Gokhale S. Generation of
iPSCs from cultured human malignant cells. Blood 2010; 115: 4039–42.
89 Mathieu J, Zhang Z, Zhou W, Wang AJ, Heddleston JM. HIF induces human
embryonic stem cell markers in cancer cells. Cancer Res 2011; 71: 4640–52.
90 Allegrucci C, Rushton MD, Dixon JE et al. Epigenetic reprogramming of
breast cancer cells with oocyte extracts. Mol Cancer 2011; 10: 7.
Dewi et al. Cancer Sci|
ª ª 2011 Japanese Cancer Association
| vol. 103| no. 3|