IJC

International Journal of Cancer

Tumor heterogeneity and cancer stem cell paradigm: Updates

in concept, controversies and clinical relevance
Anup Kumar Singh1*, Rakesh Kumar Arya1*, Shrankhla Maheshwari1,2*, Akhilesh Singh1,
Sanjeev Meena1, Priyanka Pandey3, Olivier Dormond4 and Dipak Datta1,2
1
Biochemistry Division, CSIR-Central Drug Research Institute, Lucknow, India
2
Academy of Scientific and Innovative Research, New Delhi, India
3
National Institute of Biomedical Genomics, Kalyani, West Bengal, India
4
Department of Visceral Surgery, Centre Hospitalier Universitaire Vaudois and University of Lausanne, Switzerland

Mini Review
Although tumor heterogeneity is widely accepted, the existence of cancer stem cells (CSCs) and their proposed role in tumor
maintenance has always been challenged and remains a matter of debate. Recently, a path-breaking chapter was added to
this saga when three independent groups reported the in vivo existence of CSCs in brain, skin and intestinal tumors using
lineage-tracing and thus strengthens the CSC concept; even though certain fundamental caveats are always associated with
lineage-tracing approach. In principle, the CSC hypothesis proposes that similar to normal stem cells, CSCs maintain self
renewal and multilineage differentiation property and are found at the central echelon of cellular hierarchy present within
tumors. However, these cells differ from their normal counterpart by maintaining their malignant potential, alteration of
genomic integrity, epigenetic identity and the expression of specific surface protein profiles. As CSCs are highly resistant to
chemotherapeutics, they are thought to be a crucial factor involved in tumor relapse and superficially appear as the ultimate
therapeutic target. However, even that is not the end; further complication is attributed by reports of bidirectional regenera-
tion mechanism for CSCs, one from their self-renewal capability and another from the recently proposed concept of dynamic
equilibrium between CSCs and non-CSCs via their interconversion. This phenomenon has currently added a new layer of com-
plexity in understanding the biology of tumor heterogeneity. In-spite of its associated controversies, this area has rapidly
emerged as the center of attention for researchers and clinicians, because of the conceptual framework it provides towards
devising new therapies.

The majority of tumors display substantial degree of pheno-
typic and functional heterogeneity at the population level.1–3
Heterogeneity among cancer cells arises within the same
tumor as a consequence of intrinsic, such as genetic4 and
epigenetic changes5 as well as extrinsic factors like varied
microenvironment at different spatial locations.6 Tumor cell
heterogeneity may result from either clonal evolution driven
by genetic instability and/or from differentiation of stem-like
cells often called cancer stem cells (CSCs).7 The CSC model
has rapidly emerged as the mainstream idea as it best fits
and logically explains the versatile attributes of heterogene-
ous tumor populations. CSCs are characterized by their abil-
Key words: tumor heterogeneity, cancer stem cell, plasticity
*A.K.S., R.K.A. and S.M. contributed equally to this work
Grant sponsor: CSIR (BSC106, BSC102, Fellowship Grants), MOES
(GAP0118), UGC Fellowship Grant
DOI: 10.1002/ijc.28804
History: Received 8 Oct 2013; Accepted 11 Feb 2014; Online 22 Feb
2014
Correspondence to: Dipak Datta, Biochemistry Division, CSIR-
CDRI, B.S. 10/1, Sector 10, Jankipuram Extension, Sitapur Road,
Lucknow-226031, India, Tel: 191-522-2772450 (Extn-4347/48),
Fax: 191-522-2771941, E-mail: dipak.datta@cdri.res.in
ity to seed tumor in animal hosts and to divide in
asymmetric manner to allow self renewal as well as differen-
tiation into non-CSC progeny lacking tumor initiating capa-
bilities (Fig. 1).8,9 Importantly, CSC rich tumors are
associated with aggressive disease, drug resistance and poor
prognosis.10 CSC model advocates the presence of tumori-
genic stem-like cells in heterogeneous tumors, which differ-
entiate into less-tumorigenic cancer cells to create a cellular
hierarchy.11 Moreover, the recently proposed concept of CSC
plasticity suggests that these cell populations are dynamic
and both CSCs and non-CSCs are capable of interconversion
in response to environmental cues, which further complicates
the situation in terms of generating tumor heterogeneity,
though the validity of this theory in in vivo is still controver-
sial.11–13 Despite the strong evidence supporting the CSC
model in certain cancers, it is critical to acknowledge the
various limitations associated with CSC model in its concep-
tual development such as reliability of cell surface marker
based identification of CSCs and lack of direct evidence
about their in vivo existence.7 In recent times, reports com-
bining mouse models that spontaneously generate tumors
with genetic mapping put forward strong evidence in sup-
port of CSC concept.14–16 This article will not only highlight
the current controversies in CSC concept but also emphasize

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Mini Review

Figure 1. Schematic overview of different models to explain cellular origin and maintenance of tumor along with their therapeutic interven-
tions and limitations. Stochastic model considers every cell within the tumor has equal tumorigenic potential. Classical CSC model assumes
stem-like cells are the key, which can irreversibly contribute to tumor heterogeneity by self-renewing as well as by their ability to differenti-
ate into other lineages. CSC plasticity model proposes bidirectional conversion equilibrium between CSCs and non-CSCs and emphasizes
the need for a lucrative combinatorial therapeutic approach to target CSCs as well as its biarmed regeneration mechanism.

the importance of tumor niche and various intracellular
signaling cascades in shaping up tumor heterogeneity and
CSC plasticity.
CSC Hypothesis: Concept and Controversies
Concept
In 1926, Bailey proposed that cancer was initiated and main-
tained by a small number of transformed precursor cells.17 In
late Nineties, Dick group in their pioneering studies, first
documented and characterized the existence of CSCs in
hematopoietic malignancies.18 Later, the CSC concept has
been extensively expanded to cover most of the solid
tumors.19,20 As per the CSC model, similar to their tissue of
origin, tumor cells are organized hierarchically with a subset
of cells called CSCs that are at the top of hierarchy, possess-
ing self renewal and multilineage differentiation capabilities.8
There are four key characteristics that define the CSC popu-
lation: (a) compared with other tumor cells, only a small por-
tion of the cells within a tumor have tumorigenic potential
when transplanted into immune-deficient mice; (b) it can be
separated from other cancer cells based on their distinctive
cell surface markers; (c) tumor, resulting from the CSCs, con-
tains mixed tumorigenic and nontumorigenic cells of the
original tumor; and (d) it can be serially transplanted
through multiple generations, indicating its self renewing
capabilities.21 It is noteworthy to mention that all cancers do
not necessarily follow CSC model and in such cancers,
although genetic and/or phenotypic heterogeneity are present,

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Singh et al.
they are equally tumorigenic and do not possess cellular hier-
archy in them.22,23 It has been also proposed that clonal evo-
lution model and CSC model may act together in a
cooperative manner within the same tumor to drive tumor
cell heterogeneity.24 In light of new reports about reversibility
of cancer cells,11–13 stochastic and CSC models are inter-
linked and CSC model is shifting from a rigid hierarchical to
a more flexible model to encompass a larger group of tumors
within its domain.25
Controversies
Central to the controversy over the CSC concept is the lack
of benchmark functional assays, over-reliance on cell surface
markers, inconsistencies in terminology and plasticity
between cell types. Especially in case of solid tumors, the
identification of CSCs by so called defined cell surface
markers has poor reproducibility and our tendency not to
publish data that are difficult to interpret or inconsistent
with existing model has amplified the perplexity of our
understanding.26,27 As an example, initially CD133 was pro-
posed to robustly distinguish CSCs from non-CSCs in brain28
and colon29 cancer cells but a series of subsequent studies
found that CD1332 also have similar tumorigenic potential
in vivo.30–32 Additionally, markers expressed by CSCs differ
among patients suffering from the same type of cancer. A
key question raised by such differences is whether tumors of
the same type differ in the extent to which they are hierarchi-
cally organized.27 Another important parameter is to judge
the tumorigenic potential of CSCs under in vivo condition
where it largely relies on the immune-competency potential
of the host. At least, in the case of melanoma, Quintana et al.
convincingly proved that modification of xenotransplantation
assays could dramatically increase the detectable frequency of
tumorigenic cells that are commonly known as a rare popula-
tion.23 Further controversy in CSC model exists at the level
of origin and terminology of CSCs during tumor initiation.
The term “CSC”’ could be misleading, when it implies that
such cells can only be derived from the stem cells of the cor-
responding tissue.21 CSCs may come up from normal stem
cells by mutations of specific oncogenes that make them can-
cerous, but again this may not be the case in all tumors. In
respect to the recently proposed CSC plasticity in solid
tumors, CSC may even arise from cells that are of non stem
cell, obviously put forward their stemness identity in jeop-
ardy.25 Therefore, careful optimisation of assay system and
using multiple combinations of endogenous and/or surface
markers will possibly provide a clearer picture as well help us
to identify, isolate and characterise quintessential CSCs in
particular cancer types.
In Vivo CSC Existence: Is It Resolving the Debate?
Until recently, most of the CSC literature addressed the poten-
tial of these cells to contribute to the disease, but not the
actual fate of the individual cell in real in vivo situation. In
principle, the cells that generate new tumors in transplantation
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studies are the CSCs but such tumorigenic potential can only
be tested in a permissive environment. Sceptics have persis-
tently pointed out that transplantation removes cells from
their native environment and may therefore, change their
behavior and tumorigenic potential.33 Moreover, transplanta-
tion studies do not provide any scope to test whether human
cells have tumorigenic potential if they are killed by the xen-
ogeneic immune response. Simply demonstrating the in vivo
tumorigenic and nontumorigenic capabilities of a tumor cell
to an immune-compromised animal host is not sufficient for
the CSC model and other criteria like the potential of
Mini Review
tumorigenic cells to give rise to non-tumorigenic progeny has
to be satisfied. Without demonstrating a fate map for lineage
relationship between CSCs and non-CSCs, the functional dif-
ferences between these cells may be considered as a result of
genetic differences. There are several technical challenges
associated with the study of CSC fate in vivo; such as diffi-
culty in distinguishing them from their differentiated prog-
eny, scarcity of surface markers exclusively present on CSCs
and specifically associated with its phenotype. However, with
the technical advancement, combining classical lineage-
tracing approach with marker based assays, three different
groups independently demonstrated the fate of CSCs in vivo
in tumor progression.14–16 Driessens et al., in their elegant
studies found that benign papillomas are hierarchically
organized and followed CSC model in vivo.14 Employing fate
map by selective ablation of Nestin1 CSCs in mouse model
of glioma, Chen et al. demonstrated that Nestin1 cells are
main driving force for tumor growth, therapy resistance and
tumor relapse.15 However, long term effects of selective abla-
tions of Nestin1 cells could not be assessed as mice inde-
pendently developed lethal tumors other than the original
ones. Therefore, the exact contribution of Nestin1 cells
remains unclear in terms of driving long lasting tumor
growth and its possible interrelationship with Nestin2 cells.
By using multicolour conditional reporter assay, Schepers
et al. discovered that in accordance with CSC theory, most of
the Lgr51 intestinal CSCs differentiated into Lgr52 non-
CSCs in vivo, though Lgr52 cells were not being fate mapped
in their experiment.16 Interestingly, in contrast to the previ-
ous finding, a recent study shows that Lgr52 cells can also
act as cell of origin for intestinal adenomas and seems not to
follow the CSC model.34 This counter challenging evidence
may possibly support the concept of CSC plasticity where
non-CSCs can be dedifferentiated into CSCs since Lgr52 cells
can give rise to Lgr51 cells in in vivo condition.
These reports provided a strong support about the pres-
ence of stem-like cells in heterogeneous tumor populations in
vivo and propelled the use of modern form of lineage tracing
in CSC biology; nevertheless it did not end the CSC debate
as lineage tracing has its own limitations in noisy and
dynamic tumor microenvironment.35–38 Most of the lineages
tracing approaches use DNA-label retention or inducible
genetic labelling and follow the fate of labelled cells and their
progenies using single or multicolour reporters depending on
 1994
single or multiple markers used. During lineage tracing, it is
imperative to note that DNA-label retention is not only
restricted to quiescent stem-like cells, but also a characteristic
of post-mitotic highly differentiated senescent cells. Also, it is
of utmost importance to ensure that the site of tracing initia-
tion should be carefully mapped as recombinant alleles or
transgenes are often mosaically expressed between different
group of cells.36 Another constraint of lineage tracing using
cell transplantation is that single cells may not behave in the
context of a graft as they do during normal tissue homeosta-
sis as lineage tracing in tumor epithelial cells demonstrated
Mini Review
altered differentiation pattern compared to intact tissue.37 For
example, lineage tracing in mammary and epidermal cell
transplantation studies have shown that the labelled cells
acquire multipotent phenotype though this is not observed in
the event of lineage tracing in undamaged tissues.39 High
coverage sequencing of human tumors as well as live imaging
of human tumors is emerging as alternative tools to study
the fate choice of tumor cells in driving tumor heterogeneity,
although their feasibility is yet to be tested.38
Solid Tumor Heterogeneity and CSC Plasticity
Within a solid tumor, a remarkable variability is found in the
morphological and physiological feature of tumor cell popu-
lation. Although cancer cell heterogeneity was classically pro-
posed after the seminal studies of Josh Fidler, Gloria
Heppner and others in the seventies,40,41 experimental explo-
ration of this subject had been limited by the conceptual
framework as well as by the unavailability of relevant study
tools. However, the discovery that tumor formation is
dependent on the acquisition of oncogenic mutations
changed the situation. These findings underlined the gene
centric prospective of tumor heterogeneity and is thought to
be attributed by genetic diversity resulting from clonal evolu-
tion. Moreover, dominance of gene centric views has been
challenged by the CSC hypothesis which additionally brings
non-genetic sources of phenotypic variability into the focus.
As a result of this, in recent years, tumor heterogeneity is
viewed under the glasses encompassing its genetic, epigenetic
and phenotypic heterogeneity.42
Certain cancer cells reversibly fluctuate among the states
with varying degree of competence to contribute to tumor
growth.11,43 An attractive model to explain this differential
ability of tumor cells is the newly established concept of
“CSC plasticity” under which the majority of tumor cells can
be considered as stem cells with varying degree of “stemness”
governed by microenvironmental cues and other stochastic
cell autonomous mechanisms (Fig. 1).12 Although lot of scep-
ticism persist in the terminology of tumor cell plasticity in
the published literature, CSC plasticity will be more appropri-
ate as it includes at least one or both stem-like cells out of
two inter-converting cells. Cancer cell plasticity would better
fit between the inter-converting tumor cells with less tumori-
genic potential and differing from each other by phenotypes
that are independent of stemness. In theory, CSC plasticity is
Tumor heterogeneity and CSC paradigm
a part of cancer cell plasticity but all cancer cell plasticity do
not belong to CSC plasticity.
Although, the underlying molecular mechanism for CSC
plasticity is yet unknown, conceptually it links clonal evolu-
tion and CSC models and demands that CSC hierarchy must
be seen as bidirectional balance between CSCs and differenti-
ated non-CSCs. Whether, CSC plasticity is orchestrated by
microenvironmental cues or simply a stochastic process may
depend on the type of tumor and its genetic background.25
Although lineage tracing studies provided strong evidence in
favor of the presence and differentiation of CSCs in vivo in
different solid tumors, they did not exclude the possibility
that labelled CSCs might itself arise from more differentiated
non-CSCs. It is also important to note that nearly all existing
reports about reversible transition between tumorigenic
(CSCs) and nontumorigenic (non-CSCs) states come from
studies on cell lines. As certain high passage cell lines have
been found to show endogenous phenotypic plasticity, it
would be critical to overcome this possibility by investigating
the extent of such plasticity in spontaneously arising tumors
in vivo.27 In support of in vivo CSC plasticity, Verma and
coworkers reported that glioma can be generated from termi-
nally differentiated neurons which can revert to a stem-like
state and express typical CSC markers following transforma-
tion, highlighting the pivotal role of genetic perturbations in
influencing CSC plasticity.44 More in depth in vivo lineage
tracing with multicolour labelling of non-CSCs and CSCs
may elucidate differentiation hierarchies and CSC plasticity
in future.
Factors Governing Solid Tumor Heterogeneity and
CSC Plasticity
The origin of intra-tumor heterogeneity and initial CSC gen-
eration within a tumor are still a matter of debate and
remains an enigma to researchers. There are various factors
that have significant role in maintaining CSC plasticity as
well as solid tumor heterogeneity, but their relative contribu-
tion to each of these phenomena is not clear yet. It seems
plausible that all the coordinated actions of these factors are
necessary to shape the phenotypic heterogeneity and govern
CSC plasticity.
Tumor microenvironment and CSC niche
The microenvironment within a solid tumor is not at all
homogeneous; rather it is a very complex network of versatile
factors and among the key forces in driving tumor heteroge-
neity.45 Unique combination of growth factors secreted by
tumor residing cells play a pivotal role in differentiating
CSCs and non-CSCs and vice versa and ultimately generates
complex heterogeneous tumors. Variations are also found at
the level of lymphatic vasculature, diverse oxygen supply, dif-
ferent numbers and types of stromal cells, infiltrating cells
and various composition of extracellular matrix along with
diverse physico-chemical properties. Therefore, cancer cells
within a given tumor are expected to experience a wide range
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Singh et al.
of micro-environmental cues, as for example, well oxygenated
tumor periphery versus hypoxic tumor core environment,
which would in turn translate into a series of phenotypic
changes including tumor heterogeneity and CSC plasticity.42
CSCs are believed to be frequently located at the hypoxic
core of solid tumors and by their unique adaptability to that
particular environment, pose a serious threat to the antitu-
mor effects of chemotherapeutic drugs. CSCs are not only
intrinsically programmed to fulfil their detrimental roles, but
are also orchestrated by stromal cells residing in their vicinity
in forming a CSC niche.46 As for example, IFNg inducible
chemokines and CXCL12 secreted by tumor infiltrating and
stromal cells interact with their receptors CXCR3, CXCR4
(pancreatic and prostate CSC marker) and CXCR7 and sub-
sequent signaling crosstalk may result in amplification of the
CSC loop.47,48 Interactions between CSCs and tumor endo-
thelial cells also play a major role in sequestering the angio-
genic process as documented in brain cancer where Nestin1/
CD1331 CSCs are located in direct proximity of endothelial
cells. Considering the close association between CSCs and
tumor endothelial cells, it has been proposed that newly
formed vasculature within a growing tumor can be derived
from the differentiation of CSCs implicating its multilineage
differentiation capabilities.49,50
Genetic instability/mutation
Although genetic instability is widely considered as a hall-
mark of cancer, its precise role in CSC generation and plas-
ticity is still uncertain. The notion that CSCs can recapitulate
the heterogeneity of the original tumor in transplantation
studies is based on certain surface markers only. At least, at
the level of initiation, spontaneous tumors are not similar to
experimental tumors induced by transplantation or by few
defined genetic mutations; rather it arises from a much more
complex process of somatic evolution. Acquisition of neces-
sary driving mutations is always associated with a larger
number of genetic alterations, which are usually silent in
nature.42 Hence, not every genetic difference between the
cells contributes to phenotypic variability but mutations that
are normally silent might increase the phenotypic plasticity
towards stemness and drug resistant stage via differential
gene expression. As an example, multidrug resistance pro-
teins such as ABCG2 are often mutated in their substrate
binding pocket enhancing their drug resistance effects and
favoring CSC like properties.51 Data from AML, breast, renal
and pancreatic cancer show extensive genetic heterogeneity
which would not be driven by merely differentiation of CSC
but rather every CSC could form genetically distinct tumor
population.52–56 Extensive genetic heterogeneity also provides
opportunity for these genetic changes to confer phenotypic
and functional heterogeneity among tumors.
MicroRNAs and other epigenetic factors
In the last few years, multiple miRNAs have been reported as
modulators of transcription factors involved in tumor
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development, progression, metastasis, drug resistance and
even in CSC maintenance and plasticity.57 Yu et al. were the
first to study miRNA expression in breast CSCs where they
observed significant downregulation of let-7, miR-16, miR-
107, miR-128 and miR-20b in CD441/highCD242/low cells
compared to non-CSCs.58 Seminal studies by the Struhl
group have put forward the direct evidence for the role of
miRNA(s) in regulating CSC plasticity in breast cancer
patients where the expression of miR-200 family members
are selectively downregulated during the acquisition of CSC
phenotype from non-CSCs.59,60 Indeed, miR-200b and miR-
Mini Review
200c overexpression strongly inhibited the proliferation of
CSCs and their ability to form tumors and this effect was
mediated by targeting different subunits of the polycomb
group (PcG) of proteins (Fig. 2).59–61 Stem cells rely on PcG
proteins to reversibly repress genes encoding transcription
factors required for differentiation.62 Various groups
hypothesize that the acquisition of promoter DNA methyla-
tion at these repressed genes could lock in stem cell pheno-
type to initiate abnormal clonal expansion and thereby
predispose to cancer.63 Furthermore, CDKN2A (p16) and
CDKN2D (p19) have been found to be transcriptionally
repressed by PcG proteins in human embryonic stem cells
and form polycomb repressive complex 2 (PRC2), which are
associated with nucleosomes that are trimethylated at Lys27
of histone H3 (H3K27).64 It has been observed that in case
of various cancers including breast, prostate, ovary and pan-
creas, the isolated CSC population overexpresses enhancer of
zeste homolog 2 (EZH2), one of the key members of PRC2,
responsible for trimethylating H3K27, which is essential for
maintenance of an intact CSC population.65,66 Particularly,
PRC1 acts downstream of PRC2 and recognizes the repres-
sive H3K27me3 marks and inhibits the transcription of genes,
responsible for stem cell differentiation. This effect of PRC1
in postponing the commitment of stem cells has been found
to be mediated by its BMI1 (B cell specific Moloney murine
leukemia virus integration site 1) component via interacting
with Ring1b.67 Furthermore, BMI1 and EZH2 are overex-
pressed in CSCs and found to be necessary for tumor initia-
tion of various solid tumors such as head and neck cancer
and glioblastoma.68 Recently, it has been reported that TGF-
b enhances CSC plasticity by reducing the presence of
H3K27me3 repressive histone modification at the ZEB1 (zinc-
finger E box-binding homeobox 1) promoter site and induce
non-CSC to CSC conversion in breast cancer.69,70 miR-200c
repressed the expression of BMI1, while loss of miR-200b
increased suppressor of zeste 12 homolog (SUZ12) expression
and H3K27 methylation.61 Interestingly, ectopic expression of
SUZ12 in transformed cells was able to generate CSCs.60 In
addition, miR-200c levels are regulated by a complicated loop
comprising BMI1 and ZEB1.71 These findings revealed that
miR-200 family members play an important role in regula-
tion of CSC formation and function via PcG complexes.
miR-200 is also involved in modulating the expression of
SRY (sex determining region Y)-box 2 (SOX2), Kruppel-like
 1996 Tumor heterogeneity and CSC paradigm
Mini Review

Figure 2. Schematic illustration of epigenetic regulation of CSC plasticity via polycomb repressor complexes (PRCs). Sequential events
describe miR-200 family mediated reversible regulation of PRC1 and PRC2 formation to maintain closed and open chromatin state in CSCs
and non-CSCs, respectively, to subsequently control the expression of differentiation phenotypes via p16 and p19 proteins.

factor 4 (KLF4), BMI1 and SUZ12 genes, previously described
to regulate stemness in cancer cells.61,72 Furthermore,
through unbiased miRNA expression profiling, Tang and col-
leagues recently showed that prostate CSCs selectively down-
regulate miR-34a and let-7b. In addition, miR-34a was shown
to directly target stem cell related genes like CD44 and
NOTCH, implying a crucial role of miRNAs in maintaining
the CSC phenotype by halting their differentiation.57
Signaling Events Involved in CSC Maintenance and
Plasticity
Although, CSCs are unique in their ability to generate
tumors, they also share various similarities to normal adult
tissue stem cells. CSCs often inappropriately use many signal-
ing pathways used by their normal counterpart and present a
challenge to the development of CSC specific therapies.73
Besides involving classical stem cell signaling pathways, it has
also been shown that interleukin-6 (IL-6)—Janus Kinase 1
(JAK1)—Signal transducer and activator of transcription 3
(STAT-3)/IL-6-JAK1-STAT3, Epidermal growth factor recep-
tor (EGFR) signaling and their crosstalk with stemness
related transcription factors play a pivotal role in regulating
CSC plasticity in solid tumors (Fig. 3).13,59,74
Classical stem cell signaling (Wnt, notch and hedgehog)
and crosstalk in CSC plasticity
Wnt/b-catenin, Notch and Hedgehog signaling pathways are
highly versatile, complex and found in embryonic or adult
stem cells in its normal form as well as in CSCs in subverted
form.75 Interestingly, many of the cell surface markers such
as Lgr5, CD44, CD24 and CD326, which have been used to
identify putative CSCs are Wnt targets suggesting its direct
role in the acquisition of CSC phenotype.73,76,77 Wnt/b-cate-
nin signaling transcriptionally regulates the expression of
OCT4, which is the key controller of CSC self renewal and
maintenance of stemness (Fig. 3). Wnt/b-catenin itself or by

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Figure 3. Schematic representation of signaling crosstalk in CSC plasticity. Classical stem cell specific (Wnt, Hedgehog and Notch) and
newly discovered (IL-6, EGFR and HIF) signaling pathways and their crosstalk in CSC maintenance and plasticity. BMI1 may act as a major
regulator of CSC state and intermediate protein linking various pathways, making it a potential target for future chemotherapeutic
strategies.
interacting with other downstream partners of Hedgehog sig-
naling pathway, promotes the expression of Jagged1/Jagged2
that are known ligands for Notch pathway.78 Hedgehog sig-
naling has been shown to regenerate CSCs by inducing epi-
thelial to mesenchymal transition (EMT) via up-regulation of
the transcription factor SNAIL and the concomitant down
regulation of E-cadherin.79 It has been demonstrated that
Hedgehog stimulation in culture increased the number and
size of mammospheres whereas, cyclopamine (a Hedgehog
pathway inhibitor) had the opposite effect indicating the
direct relationship between Hedgehog pathway and CSC gen-
eration.80 The transcriptional program orchestrated by
Hedgehog signaling depends on the Gli family of transcrip-
tion factors.81 Although Gli proteins can be converted to
either transcriptional activators or truncated transcriptional
repressors, Hedgehog-Gli interaction ultimately enhances self
renewal properties and CSC plasticity either by the direct
binding of Gli activator (GliA) to the promoters of SNAIL,
CYCLIN D, MYC and JAGGED2 etc. or via activating the key
stemness regulator BMI1 through GliR (Fig. 3).82–84 BMI1
plays a central role in the crosstalk of classical stem cell path-
ways and finally results in the maintenance and acquisition
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of CSC phenotype either by inhibiting p16 and p19 transcrip-
tion or by other unidentified functions.85
Novel role of IL-6 and EGF signaling in CSC plasticity
Although CSC plasticity is a reversible phenomenon, the rate
of interconversion between CSCs and non-CSCs differ signifi-
cantly. It has been demonstrated that CSCs rapidly differenti-
ate into non-CSCs, but non-CSCs are not easily convertible
into CSCs. This nonreciprocal conversion process between
non-CSCs and CSCs should result in the ever decreasing
number of CSCs, which is paradoxical, as CSCs are main-
tained at a constant proportion within a tumor.13 This obser-
vation led to the foundation that CSCs and non-CSCs
interact with each other in a paracrine fashion and maintain
a dynamic equilibrium in a heterogeneous tumor population
that do not occur when cells are cultured separately. In the
search of paracrine factors, it has recently been shown that
IL-6 plays a crucial role in maintaining CSC plasticity. As its
downstream target, IL-6 regulates CSC-associated OCT4,
SOX2 and NANOG gene expressions through the IL-6-JAK1-
STAT3 signal transduction pathway (Fig. 3), which converts
non-CSCs into CSCs.74 Transcription factor STAT3 is
 1998 Tumor heterogeneity and CSC paradigm

activated through phosphorylation of tyrosine residue 705
and results in nuclear translocation and activation of the
STAT3 transcriptional regulatory function.86,87 Interestingly,
STAT3 activation is mediated by members of the JAK family
proteins as well as receptor tyrosine kinase (RTK) family pro-
teins via EGFR.88 EGF-RTK signaling pathway plays a critical
role in regulating CSC state via its multiarmed downstream
cascades and acts as a link for crosstalk between Hedgehog
and IL-6 signaling. PI3K activated via EGFR signaling can
either turn on GliA via PI3K-AKT-mTORC1-S6K pathway to
induce the expression of CSC specific genes or ligands for
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development of new therapeutic strategies considering CSCs
and its relationship with tumor microenvironment are
urgently required as tumor milieu is believed to be the key
source for cancer cell differentiation and bidirectional conver-
sion.25,94 Therefore, identification of factors responsible for
bidirectional conversion of tumor cells will be of fundamental
importance.95 Keeping the newly discovered concept of CSC
plasticity in mind, effective cancer therapy should not only
target CSCs but also prevent CSC generation from non-CSCs
(Fig. 1) as CSCs can be recreated from bulk non-CSC popu-
lation at any stage of tumorigenesis. In this respect, the lead-

other pathways (Jagged 2),89 or it can activate STAT3 via
Bone marrow X-linked protein to express the stemness spe-
cific genes like OCT4, SOX2, CD44, etc (Fig. 3).90,91 Studies
have suggested that the core hypoxic zone of solid tumors
harbors highly resistant tumor cells overexpressing CSC
markers. Hypoxic condition within a tumor can induce stem-
ness in tumor population via transcriptional induction of
hypoxia inducible factor (HIF)1a and HIF2a.92 HIF1a can
also be activated via PI3K-AKT to induce EMT modulator
Twist1 and polycomb repressor BMI1 to help the downregu-
lation of p16 and p19 that are required for the differentiation
of CSCs. Unlike HIF1a, HIF2a acts as a transcriptional acti-
vator of stemness specific genes OCT4, SOX2, CD44, KLF-4,
MYC, etc.93 Hence, it is noteworthy that these newly discov-
ered signaling pathways act in synergistic manner with an
overall goal of inducing stemness and further elucidation of
their complex crosstalk will help in better understanding of
CSC plasticity.
Therapeutic Potential and Concluding Remarks
Although intermittent improvements are being made, tumor
relapse and treatment resistance remains a major cause of
morbidity and mortality that are proposed to be particularly
due to the persistence of highly resistant CSC population
during chemotherapy at least in the case of cancers following
CSC model. Hence, for complete eradication of cancer cells,
ing candidate drug is the ionophore antibiotic salinomycin
and a well known type 2 diabetic drug metformin, which
have recently been documented to successfully eliminate
CSCs in different types of human cancers in vitro and in vivo
and are also being highly effective while used in combina-
tions with other conventional chemotherapeutic drugs.96–101
Recently, pharmacological inhibitors of S phase kinase associ-
ated protein 2 (SKP2) have been demonstrated to effectively
inhibit CSCs and considered for their therapeutic poten-
tial.102,103 However, as SKP2 is also involved in general
metabolism, clinical efficacy and toxicological concerns of its
inhibitors need to be further tested. In cancers that do not
follow the CSC model, it would be sensible to avoid unusual
highlighting of the CSC theory towards drug development by
focusing on small population of cancer cells as they do not
have specific capacity to drive disease progression or tumor
relapse. Further investigation of molecular mechanism of
CSC plasticity, understanding the mode of action of CSC spe-
cific drugs and their clinical efficacy are required to offer
highly effective therapies for patients in all stages of cancer.
Acknowledgements
The authors are grateful to Dr. David M Briscoe for reading the manuscript
and his thoughtful insight. Institutional (CSIR-CDRI) communication num-
ber for this article is 8623. DD dedicates this manuscript to his beloved
teacher Dr. Samar Chakraborty.

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