Review Articles

OMICS A Journal of Integrative Biology

Volume 20, Number 12, 2016
Mary Ann Liebert, Inc.
DOI: 10.1089/omi.2016.0152

Cancer Stem Cell Hypothesis for Therapeutic

Innovation in Clinical Oncology?
Taking the Root Out, Not Chopping the Leaf
Kevin Dzobo,1,2 Dimakatso Alice Senthebane,1,2 Arielle Rowe,1,2 Nicholas Ekow Thomford,3
Lamech M. Mwapagha,1,2 Nasir Al-Awwad,4 Collet Dandara,3 and M. Iqbal Parker1,2

Abstract

Clinical oncology is in need of therapeutic innovation. New hypotheses and concepts for translation of basic
research to novel diagnostics and therapeutics are called for. In this context, the cancer stem cell (CSC)
hypothesis rests on the premise that tumors comprise tumor cells and a subset of tumor-initiating cells, CSCs, in
a quiescent state characterized by slow cell cycling and expression of specific stem cell surface markers with the
capability to maintain a tumor in vivo. The CSCs have unlimited self-renewal abilities and propagate tumors
through division into asymmetric daughter cells. This differentiation is induced by both genetic and environmental factors. Another characteristic of CSCs is their therapeutic resistance, which is due to their quiescent
state and slow dividing. Notably, the CSC phenotype differs greatly between patients and different cancer types.
The CSCs may differ genetically and phenotypically and may include primary CSCs and metastatic stem cells
circulating within the blood system. Targeting CSCs will require the knowledge of distinct stem cells within the
tumor. CSCs can differentiate into nontumorigenic cells and this has been touted as the source of heterogeneity
observed in many solid tumors. The latter cannot be fully explained by epigenetic regulation or by the clonal
evolution theory. This heterogeneity markedly influences how tumors respond to therapy and prognosis. The
present expert review offers an analysis and synthesis of the latest research and concepts on CSCs, with a view
to truly disruptive innovation for future diagnostics and therapeutics in clinical oncology.
Keywords: cancer stem cells, biomarkers, tumour initiating cells, heterogeneity, stem cell markers, therapeutics, disruptive innovation, innovation systems
Introduction

here is a need for new hypotheses and conceptual approaches for discovery and translational research on novel
diagnostics and therapeutics in support of precision medicine
in clinical oncology (Ren et al., 2014). The heterogeneity observed in solid tumors has traditionally led scientists to speculate that there is a hierarchical organization within tumors
where some cells are less differentiated than others (Furth et al.,
1937; Hewitt, 1958; Lapidot et al., 1994; Makino, 1956). The
idea of less differentiated cells in tumors gave birth to the
concept of tumor-initiating cells (TICs) (Hermann et al., 2007).

Through several studies, scientists were able to show that

starting with a single cell, it is possible to propagate tumor
xenographs with similar characteristics to the original tumor
(Clarkson and Fried, 1971; Kleinsmith and Pierce, 1964;
Pierce and Wallace, 1971). This is the premise on which the
cancer stem cell (CSC) theory as hypothesized by Pierce in 1971
rests (Pierce and Wallace, 1971). However, more scientific
discoveries of oncogenes and tumor-suppressing genes opposed the CSC theory (Clarkson and Fried, 1971; Fearon
and Vogelstein, 1990; Nowell, 1976). It was shown, for
example, tumors originated from a single cell that acquired
mutations giving the tumor cell an advantage over normal

1
International Centre for Genetic Engineering and Biotechnology (ICGEB), Cape Town Component, Wernher and Beit Building (South),
UCT Medical Campus, Anzio Road, Observatory 7925, Cape Town, South Africa.
2
Division of Medical Biochemistry and Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical
Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa.
3
Pharmacogenetics Research Group, Division of Human Genetics, Department of Pathology and Institute of Infectious Disease and
Molecular Medicine, Faculty of Health Sciences, University of Cape Town, South Africa.
4
Department of Clinical Pharmacy, Faculty of Clinical Pharmacy, Albaha University, Albaha, Saudi Arabia.

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cells. This is termed the clonal evolution theory as hypothesized by Nowell in 1976 (Nowell, 1976). The clonal evolution
theory, however, failed to explain the heterogeneous nature of
some tumors and why tumors recur after periods of dormancy.
Further studies focusing on leukemia sparked renewed interest in the CSC theory after it was shown that there is so
much heterogeneity in leukemia and that there are specific
cell surface markers that differentiate the tumorigenic cancer
cells from the nontumorigenic cancer cells (Bonnet and Dick,
1997; Spangrude and Scollay, 1990; Spangrude et al., 1988;
Szilvassy et al., 1990).
That no tumor is similar to another has been proven beyond
any doubt and this is partly due to different genetic mutations
and the contribution of the tumor microenvironment (Dalerba
and Clarke, 2007; Kreso and Dick, 2014; Pece et al., 2010;
Stingl and Caldas, 2007; Visvader and Lindeman, 2012).
Several studies show that CSCs play a crucial part in tumor
initiation and development (Bonnet and Dick, 1997; Stingl
and Caldas, 2007). According to the CSC model, a portion of
the stem cells is responsible for maintaining tumorigenesis
and results in the heterogeneity found in many tumors
(Hermann et al., 2007; Kreso and Dick, 2014; Pece et al.,
2010; Visvader and Lindeman, 2012). The source of CSCs is
plagued with controversy (Clarke and Fuller, 2006; Kim
et al., 2005; Visvader and Lindeman, 2012).
Although CSCs have the ability to self-renew and differentiate, it is known that they can also originate from normal
cells other than adult stem cells (Clarke and Fuller, 2006;
Kreso and Dick, 2014; Stingl and Caldas, 2007; Visvader and
Lindeman, 2012). CSCs have been identified in many types of
tumors, although they might behave differently in different
tumors (Clarke and Fuller, 2006; Hermann et al., 2007; Pece
et al., 2010; Stingl and Caldas, 2007; Tirino et al., 2008).
Depending on the source of information, CSCs can also be
called TICs. CSCs are distinguished from nontumorigenic
cancer cells by two main properties: their ability to self-renew
and their ability to differentiate into many cell types, meaning
they can initiate or maintain a tumor (Bonnet and Dick, 1997;
Lapidot et al., 1994; Visvader and Lindeman, 2012).
The idea that cancers arise from stem cells did not start
with the CSC theory. Before the CSC theory was the embryonal rest theory of cancer (Conheim, 1875; Durante,
1874). This theory suggested that cancers are a result of
remnants of embryonic tissue, pluripotent stem cells, and
signaling from early development that acquired malignant

DZOBO ET AL.

properties and can become tumorigenic (Conheim, 1875;

White and Lowry, 2015). This theory came from observations that cancers were associated with embryonic tissue/cells
that have been activated to become tumors. Normally these
embryonic tissues/cells are dormant. This theory, however,
was generally discredited back then.
The CSC Model

There are two hypotheses that attempt to explain the intratumoral heterogeneity as seen in solid tumors (Hamburger
and Salmon, 1977; Nowell, 1976). The first hypothesis to be
proposed is the clonal evolution theory, which states that
most tumors originated from a single cell (Nowell, 1976).
According to the clonal evolution theory, the development of
a tumor is therefore a result of an accumulation of mutations
within the original clone. The second hypothesis is the CSC
model, which proposes that CSCs are a small subset of the
tumor, but have the ability to self-renew and start new tumors
(Bonnet and Dick, 1997; Lapidot et al., 1994; Rocco et al.,
2012; Thenappan et al., 2009) (Fig. 1).
Each of these hypotheses has its merit. Through the use of
flow cytometry, it was shown that some human acute myeloid
leukemias and colon, pancreatic, brain, ovarian, and breast
cancers follow the CSC model (Bonnet and Dick, 1997; Lapidot
et al., 1994; Li et al., 2007; Piccirillo et al., 2006; Tominaga
et al., 2016). This strongly implies that some cancers are organized into tumorigenic and nontumorigenic components
(Bonnet and Dick, 1997; Lapidot et al., 1994; Magee et al.,
2012). The nontumorigenic cells can be of different phenotypes.
The CSC model is largely dependent on the isolation of
TICs and transplantation limiting dilution assays (Behnan
et al., 2016; Islam et al., 2015; Leon et al., 2016; Moghbeli
et al., 2014). The CSC model is also dependent on the use of
cell surface markers for the identification of cells (Bonnet and
Dick, 1997; Clarke and Fuller, 2006; Lapidot et al., 1994;
Quintana et al., 2008). CSCs have been isolated using cell
sorting technologies that use antibodies against various surface
markers such as CD44, CD24, CD133, ALDH1, and CD166
(Al-Hajj et al., 2003; Kim et al., 2005; Prince et al., 2007;
Ricci-Vitiani et al., 2007; Schatton et al., 2008; Singh et al.,
2003; Szotek et al., 2006) Surface markers, however, do not
adequately differentiate CSCs from non-CSCs. Of late, studies
have shown that CSCs are a tumorigenic reservoir that can
initiate relapse in certain cancers (Dalerba and Clarke, 2007;

FIG. 1. The CSC Model. Solid tumors comprise both tumorigenic cancer cells (therefore CSCs) (red) and nontumorigenic cancer
cells (green and blue). The tumorigenic cells and therefore CSCs, normally a minority, can divide into cancer cells of different
phenotypes. The tumorigenic cells give rise to the phenotypic heterogeneity seen in many solid tumors. CSC, cancer stem cell.

CANCER STEM CELLS AND THERAPEUTIC INNOVATION

Hermann et al., 2007; Tirino et al., 2008). However, the observation that cancers driven by CSCs such as in the case of
leukemia also show clonal evolution when tyrosine kinase inhibitors are used in treatment illustrates that the clonal evolution model and the CSC model are not exclusive (Pece et al.,
2010; Stingl and Caldas, 2007; Visvader and Lindeman, 2012).
Evaluation of the existence of CSCs started through the
use of cultured tumor cells (Bonnet and Dick, 1997;
Hermann et al., 2007; Kemper et al., 2010; Kreso and Dick,
2014). However, the approach has shifted with the use of tumor
samples that are freshly obtained and less passaged cancer cells
for transplantation studies being considered the norm (Dalerba
and Clarke, 2007; Fargeas et al., 2003; Lathia et al., 2011; Pece
et al., 2010). This review is an attempt to highlight developments in the field of CSCs. We attempt to show the diversity
involved in CSC pools and the possibility of non-CSCs obtaining the CSC-like phenotype. This has huge implications on
therapeutic strategies to treat cancer as all CSC pools within the
tumor will have to be taken into consideration when designing
drugs. Therefore, future therapies will have to target both CSCs
and non-CSCs for effective treatment of cancer.
CSC Markers

CSC markers are not universal for all cancer types. In different cancers, a variety of markers have been used to isolate
and identify subsets enriched for CSCs and these include
CD44, CD24, CD 133, EpCAM, and ALDH activity (Dalerba
and Clarke, 2007; Hermann et al., 2007; Ko et al., 2013; Kreso
and Dick, 2014; Read et al., 2009; Scatena et al., 2013; Suszynska et al., 2014; Tirino et al., 2008; Verma et al., 2016;
Visvader and Lindeman, 2012). Surface markers such as CD44
and CD24 are the most useful markers for isolation of subsets
enriched for CSCs (Kim et al., 2005, 2016; Leon et al., 2016;
MacDonagh et al., 2016; Prince et al., 2007; Schatton et al.,
2008; Szotek et al., 2006; Yang and Rycaj, 2015).
However, these markers are also expressed by other cells and
are not exclusively expressed by CSCs (Du et al., 2016). It has
been observed that the CSC phenotype differs from patient to
patient even for the same tumor type (Kreso and Dick, 2014;
Lathia et al., 2011; Singh et al., 2004; Ward et al., 2009). Many
of these markers are modulated or controlled by extrinsic factors. For example, CD133 has been reported to be specifically
expressed by tumorigenic brain cancer cells, yet further studies
showed that both CD133+ and CD133- brain tumor cells can
form tumors (Beier et al., 2007; Joo et al., 2008).
Additional CSC markers for specific tumors such as brain
tumors are SSEA-1, CD15, and a6 integrin (Lathia et al., 2011;
Son et al., 2009; Visvader and Lindeman, 2012). The best way
to refine the CSC phenotype is through the use of a combination
of markers. Differences observed in terms of CSC markers
could also be due to differences among cancer patients and
different methods used in the isolation of CSCs. It has also been
observed that some markers such as CD133 are diminished in
some cancers during passaging (Stewart et al., 2011).
Beside the use of antibodies to isolate CSCs based on cell
surface markers, another strategy employed by many researchers to isolate CSCs involves the use of side population
cells. Side population cells in tumors are a small subpopulation of cancer cells with stem cell-like properties and can be
isolated and identified by dual-wavelength FACS analysis
(Zhang et al., 2012; Zhao et al., 2014). The problem with

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FACS is that the recovered cells are usually of low viability

and it requires cell suspensions and not solid samples. In
several cancers, it has been shown that side population tends
to enrich CSCs. Stem cell-like subpopulations can be isolated
from various cell lines through the use of the Hoechst 33342
dye (Dieter et al., 2011; Haraguchi et al., 2006). Side populations have been isolated from several solid tumors as well
and have been shown to have stem cell-like properties.
Major disadvantages of using side population include low
specificity and the lack of purity of cells obtained. In addition,
Hoechst 33342 is very toxic to cells. Yet another strategy to
enrich CSCs is the use of sphere formation. Sphere formation
is useful to enrich the potential CSC population as a functional approach. Tumor sphere-forming cells are known to
express several stem cell markers such as CD44 and ALDH
(Zhang et al., 2012; Zhao et al., 2014). The tumor sphere is
considered a valuable model for the study of CSCs and
chemoresistance. Magnetic-activated cell sorting (MACS) is
yet another method used for CSC isolation. In this method,
microbeads are linked to a monoclonal antibody that allows
the separation of cells based on the expression of a specific
antigen. MACS is very fast and easy to use, but requires cell
suspensions and cannot be used with solid samples.
Heterogeneity in CSC Phenotype and Metastasis

Many reasons have been put forward to explain the presence

of different phenotypes of CSCs from the same tumor and
these include the presence of different CSC pools, the enzymatic methods used in the isolation of CSCs, and also whether
the CSCs are obtained from cultured or freshly sorted cells
(Curtis et al., 2010; Dalerba and Clarke, 2007; Ginestier et al.,
2007; Hermann et al., 2007; Meyer et al., 2010; Stingl and
Caldas, 2007). The idea that CSCs can interconvert between
stem and nonstem cells adds another layer of complexity to the
two models put forward, CSC and clonal evolution models, but
might be the reason why there is tumor heterogeneity in solid
tumors (Chen et al., 2010; Goardon et al., 2011; Kreso and
Dick, 2014; Pece et al., 2010; Schober and Fuchs, 2011;
Stewart et al., 2011; Tirino et al., 2008).
The interconversion of CSCs into nontumorigenic cells
generates phenotypic and functional heterogeneity (Dick,
2008; Islam et al., 2015; Kise et al., 2016; Magee et al., 2012;
Moghbeli et al., 2014; Shackleton et al., 2009; Yang and
Rycaj, 2015). Microenvironmental cues might influence the
behavior of CSCs and nonstem cells as well. Such microenvironmental cues include autocrine and paracrine factors,
hypoxia, the extracellular matrix, and nutrient supply. Thus,
CSCs and nonstem cells are adaptable cells capable of
changing their states and are therefore considered to be
plastic (Kise et al., 2016; Malanchi et al., 2012; Moghbeli
et al., 2014; Pece et al., 2010; Stingl and Caldas, 2007; Visvader and Lindeman, 2012; Wang et al., 2010).
The presence of CSCs and/or TICs in many solid tumors is
infrequent and these cells show greater resistance to commonly used chemo- and radiotherapies than differentiated
tumor cells (Chen et al., 2010; Kise et al., 2016; Kreso and
Dick, 2014; Stewart et al., 2011; Stingl and Caldas, 2007;
Visvader and Lindeman, 2012). Cells that are able to initiate
the growth of tumors after transplantation are defined as
TICs. CSCs are not always a minor component of a tumor. An
increased pool of CSCs has been associated with aggressive

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high-grade tumors (Chen et al., 2010; Clarke and Fuller,

2006; Hermann et al., 2007; Pece et al., 2010). TICs isolated
from primary cancer cells and from xenografts do not necessarily express the same markers (Behnan et al., 2016; Boiko
et al., 2010; Dalerba and Clarke, 2007; Kise et al., 2016;
Meyer et al., 2010; Stewart et al., 2011; Verma et al., 2016).
Thus, the CSC phenotype may be altered by the CSC surrounding environment or niche and this might explain the
origin of the heterogeneity within the TICs (Behnan et al.,
2016; Boiko et al., 2010; Dalerba and Clarke, 2007; Kise
et al., 2016; Meyer et al., 2010; Stewart et al., 2011).
Cancers are known to have distinct populations of CSCs. It
is also known that cancers may have CSC pools that recapitulate the immunophenotype of primary cancer cells
(Stewart et al., 2011; Visvader and Lindeman, 2012). This
means that CSCs can dedifferentiate or can convert into a
different CSC. Different oncogenic mutations can give rise to
cancers, differing in the way they follow the CSC model and
in tumorigenic cell phenotype. Limited self-renewal abilities
have been observed in some cancer cells capable of initiating
tumors in colorectal cancers (Du et al., 2016).
In some cases, latent CSCs have been characterized and
found to be able to self-renew and initiate tumors after several
transplantation assays (Dieter et al., 2011; Kise et al., 2016;
Kreso and Dick, 2014). Two different CSC pools in skin
squamous cell carcinomas have been shown to interchange
phenotype and showed different tumor growth kinetics
(Schober and Fuchs, 2011). Many studies have shown that
genetic signatures in CSCs may predict their ability to form
metastases (Pallini et al., 2008; Shiozawa et al., 2013; Woo
et al., 2011). The expression of CD133, one of the CSC
markers, has been correlated with cancer cell proliferation
and patients survival in oligodendroglia tumors and rectal
cancer (Beier et al., 2008; Wang et al., 2009). Another strong
predictor of tumor recurrence in colorectal cancer is the
methylation of Wnt target gene promoters (de Sousa et al.,

DZOBO ET AL.

2011; Shiozawa et al., 2013). Many studies assume that CSC

characteristics are universal without the need for validation to
other patients, cell lines, and to cells transformed in culture.
A growing body of data shows that there is a subset of
tumor cells that impart metastatic activity (Dalerba and
Clarke, 2007; Hermann et al., 2007; Kise et al., 2016; Liu
et al., 2010). Potentially, cells within the CSC subsets may
have metastatic capacity. This metastatic capacity may be a
result of epithelialmesenchymal transition occurring at the
invading edge of the tumor. The origin of the metastatic
CSCs may be varied and ranges from the primary tumor
CSCs to non-CSCs within the tumor (Fig. 2). The relationship
between CSCs and metastasis has not been resolved to date
and most studies examine cancer cells found in circulation,
focusing on their tumorigenic potential. Several researchers
have suggested that CSCs gain their metastatic potential
through the epithelial-to-mesenchymal transition (EMT)
process (Hsu et al., 2016; Kalluri and Weinberg, 2009;
Verma et al., 2016).
In most tissues, the process of EMT allows cells to move
from one location to another. The same process can be activated by CSCs to allow these cells to migrate from one location to another (Peter, 2010; Shiozawa et al., 2013). It is also
thought that cues from the CSC niche may induce EMT in
CSCs (Armstrong et al., 2011; Kalikin et al., 2003; Shiozawa
et al., 2013). Midbody, a protein structure needed in separating
cells during cell division, has been seen to accumulate in
cancer cells and is thought to increase the tumorigenicity of
cancer cells (Kuo et al., 2011; Shiozawa et al., 2013).
Not all cancers have CSCs (Clarke and Fuller, 2006; Kise
et al., 2016; Stingl and Caldas, 2007; Visvader and Lindeman, 2012). A tumor is more than just a mass of cancer cells
and CSCs. Cancers comprise the associated stroma, hematopoietic cells, inflammatory cells, and the vasculature (Dalerba and Clarke, 2007; Kise et al., 2016; Kreso and Dick,
2014; Pece et al., 2010). It is obvious that CSCs require a

FIG. 2. CSC involvement in metastasis. Several theories have been put forward to explain the involvement of CSCs in
metastasis. The primary tumor contains cancer cells, CSCs, and normal tissue cells. (1) CSCs can metastasize to distant
organs; (2) CSCs undergo EMT and circulate the body through the blood or the lymphatic system; (3) Both cancer cells and
CSCs can metastasize to distant organs. EMT, epithelial-to-mesenchymal transition.

CANCER STEM CELLS AND THERAPEUTIC INNOVATION

specific microenvironment to be able to self-renew and differentiate (Beck et al., 2011; Gilbertson and Rich, 2007;
Wang et al., 2010). The relationship between all cells, including CSCs, and the local environment is bidirectional. The
cellular niche has been shown to alter the behavior of different cells, including CSCs (Clarke and Fuller, 2006; Dzobo
et al., 2015, 2016; Heddleston et al., 2009; Hjelmeland et al.,
2011; Kise et al., 2016; Visvader and Lindeman, 2012). CSCs
are also known to secrete growth factors that can alter the
environment and encourage tumor vasculature growth
(Clarke and Fuller, 2006; Gilbertson and Rich, 2007; Hermann et al., 2007). Microenvironmental cues play a critical
part in deciding the fate of many cells, including CSCs (Beck
et al., 2011; Dzobo et al., 2015, 2016; Gilbertson and Rich,
2007; Wang et al., 2010). A lot of effort is therefore being
directed at the CSC niche as a therapeutic target (Beachy
et al., 2004; Beck et al., 2011; Calvi et al., 2003; Dalerba and
Clarke, 2007; Pece et al., 2010).
Many cells within the tumor microenvironment such as
myofibroblasts play significant roles in modulating CSC
status in several tumors (Kreso and Dick, 2014; Vermeulen
et al., 2010; Visvader and Lindeman, 2012; Wei et al., 2008).
CSCs are known to secrete IL6 to attract cells such as mesenchymal stem cells (MSCs) to the tumor. In turn, MSCs
upregulate signaling pathways, such as NF-kb and Wnt,
necessary for stemness maintenance in CSCs. TGF-b is also
known to interact with NF-kb signaling to promote cancer
stemness. Thus, the tumor microenvironment can govern
tumor cell stemness. Therefore, when it comes to the CSC
phenotype, there are niche-induced changes and these point
to the tumor stroma as being a potential target to eventually
kill CSCs (Vermeulen et al., 2010; Wei et al., 2008).
CSCs can also hijack the normal stem cell niche established by cells such as fibroblasts and MSCs. This niche is
rich in factors such TNF-a and TGF-b and these enhance the
stemness of CSCs and promote EMT. Cancer-associated fibroblasts produce matrix metalloproteases and these can
promote ECM remodeling, thereby promoting EMT and CSC
stemness. Recent studies have focused on the tumor stroma
and its possible role in tumor progression (Dzobo et al., 2015,
2016; Malanchi et al., 2012; Raaijmakers et al., 2010; Vermeulen et al., 2010; Wei et al., 2008). These studies have
attempted to include tumor stroma components, cells, and the
ECM in their experimental setups so as to recapitulate the
in vivo tumor environment (Dzobo et al., 2015, 2016; Malanchi et al., 2012; Raaijmakers et al., 2010).
In addition, several cancers have been associated with inflammatory diseases (Cabodi and Taverna, 2010; Greer and
Whitcomb, 2009). This is partly because most carcinogenic
agents and viruses are known to induce inflammation (Orr
et al., 2013; Schwitalla et al., 2013). Many solid tumors are
associated with elevated levels of reactive oxygen species and
inflammation (Schetter et al., 2010). Inflammation results in
the release of many factors such as growth factors and cytokine
signaling molecules and these modify the tumor microenvironment and affect/transform especially cells such as fibroblasts, macrophages, neutrophils, and lymphocytes. Most
importantly, cytokines can activate signaling pathways required by CSCs to undergo EMT. Whether inflammation is
pro- or antitumor depends on many factors such as the abundant cell type within the tissue, the degree of inflammation,
and the extracellular matrix (Grivennikov et al., 2010).

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The necessity of inflammation during tumor initiation and

progression is, however, not clear. Several studies have suggested that chronic inflammation can alter or reprogram cell
fate and can impart stem cell-like properties on differentiated
cells (Orr et al., 2013; Schwitalla et al., 2013; Song and Balmain, 2015; White and Lowry, 2015). Several reports have
shown that inflammation can play a part in the dedifferentiation
of several cell types. For example, activation of NF-jb, a
proinflammatory nuclear factor, has been shown to increase
the stimulation of oncogenic Wnt through the stabilization of
b-catenin (Schwitalla et al., 2013). This resulted in increased
downstream Wnt signaling and the dedifferentiation of differentiated villus cells (Schwitalla et al., 2013; White and
Lowry, 2015). Furthermore, many properties of a tumor such as
invasiveness and metastatic aggressiveness are all linked to the
degree of inflammation (Sparmann and Bar-Sagi, 2004, 2005).
CSCs and Therapy

Current therapies fail to prevent cancer relapse and metastasis partly due to the presence of therapy-resistant tumor
stem cells (Cabrera et al., 2015; Chen et al., 2010; Devesa
et al., 1998; Gangopadhyay et al., 2013; Hsu et al., 2016;
Lawrence et al., 2015; Maugeri-Sacca et al., 2011; Nie et al.,
2015; Rice et al., 1997; Shen et al., 2016; Yi et al., 2013).
Many studies focused on the biochemical and genetic involvement in CSC drug resistance (Kreso and Dick, 2014). In
several studies, one of the major mechanisms for posttherapeutic recurrence of esophageal, colorectal, and breast
cancer has been suggested to be through CSCs (Castagnoli
et al., 2016; Islam et al., 2015; Masuda et al., 2016; MaugeriSacca et al., 2011; Rice et al., 1997; Shen et al., 2016; Tominaga et al., 2016). Resistance to current therapies has been
used to define CSCs, but the sensitivity of TICs and nontumorigenic cells depends on the type of cancer and the
therapy used (Gedye et al., 2016; Kim and Dirks, 2008;
Najumudeen et al., 2016) (Fig. 3).Therapy resistance has also
been known to arise from genetic mechanisms not related to
CSCs. CSCs have been noted for their increased efflux capacity especially that of drugs.
Several membrane transporter proteins known as the ATPbinding cassette (ABC) proteins are known to play critical
roles in CSC drug resistance through promotion of drug efflux (Di and Zhao, 2015). ABC proteins are implicated in the
development of multidrug resistance and this gives CSCs the
ability to resist many therapeutic drugs. Chief among these
ABC transporters are the ABCB1, ABCC1, and breast cancer
resistance protein (BCRP) (Di and Zhao, 2015). ABCB1, also
known as P-glycoprotein, is normally expressed at low levels
in normal cells and plays a role in transporting toxins and
xenobiotics out of the cell. In many cancers, including lung,
liver, prostate, brain, and ovarian cancers, there is an observed increase in the expression of the ABC proteins.
Many studies have shown that the overexpression of BCRP
and ABCB1 in tumors correlates with poor response to drug
treatment (Tannock et al., 2002). In addition, chemotherapeutic drugs induce DNA lesions and these result in activation
of DNA damage response pathways in cancer cells. Several
studies have shown that CSCs can activate checkpoint response through several proteins, including checkpoint kinases
1 and 2 (Bao et al., 2006; Venkatesha et al., 2012). In many
cancers, including pancreatic, liver, and glioblastoma, CSCs

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DZOBO ET AL.

FIG. 3. Solid tumor response to conventional chemotherapy and CSC-targeted therapy. Conventional chemotherapy kills most
cancer cells within the solid tumor with the exception of CSCs. The CSCs survive and give rise to new and chemoresistant tumors.
A combination of both conventional and CSC-targeted therapies can eradicate all cancer cells, resulting in tumor shrinkage.
show increased expression of DNA repair-associated genes
such as the BRCA1 (Chen et al., 2016b; Liu et al., 2006;
Mathews et al., 2011). Another important characteristic of
CSCs is their ability to evade programmed cell death. Many
antiapoptotic genes such as Bcl-2 and Bcl-xL are reported to be
overexpressed in many cancers (Chen et al., 2016b; Furnari
et al., 2007; Hata et al., 2015; Tagscherer et al., 2008).
For novel and alternative therapies to be developed, there
is need to elucidate the mechanisms involved in controlling
self-renewal and differentiation of CSCs. Many signaling
pathways appear to be common in both CSCs and normal
stem cells (Dzobo et al., 2015; Hsu et al., 2016; Jamieson
et al., 2004; Krivtsov et al., 2006; Leon et al., 2016; Pece
et al., 2010; Shen et al., 2016; Wang et al., 2010). These
pathways include Wnt, Notch, NF-kb, JAK/STAT, PTEN,
and the Hedgehog (Fig. 4). The gene signature activated
during self-renewal in cells, including CSCs and normal
cells, appears to be different in some ways and this is good
news for drug discovery (Chen et al., 2016a; Cordenonsi
et al., 2011; Malanchi et al., 2008; Zhao et al., 2009). Recent
data suggest that self-renewal pathways are important in
maintaining CSCs and targeting such pathways might be one
of the most effective strategies available to eradicate the
CSCs (Chen et al., 2016a; Dalerba and Clarke, 2007; Leon
et al., 2016; Pece et al., 2010; Shen et al., 2016).
Many studies have focused on signaling pathways such as
the Notch/Jagged signaling pathway as a possible source of
therapeutic targets for cancer (Li et al., 2014; Wang et al., 2014;
Wei et al., 2014). The Notch/Jagged 1 signaling pathway is
known to play a significant role in tumorigenesis, cancer cell
proliferation, metastasis, and drug resistance. The Notch/Jagged 1 signaling pathway has also been shown to be required for
cancer renewal (Li et al., 2014; Wang et al., 2014; Wei et al.,
2014; Yabuuchi et al., 2013; Zhao et al., 2011). Notch/Jagged1
receptors are commonly overexpressed in many tumors (Li
et al., 2014; Wang et al., 2014; Wei et al., 2014; Yabuuchi et al.,
2013; Zhao et al., 2011). The Notch/Jagged 1 signaling has
been shown to be important in both CSC maintenance and selfrenewal (Li et al., 2014; Wang et al., 2014; Wei et al., 2014;
Yabuuchi et al., 2013; Zhao et al., 2011). Notch 1 activity is
known to be highly enriched in human breast CSC populations

and the inhibition of Notch signaling resulted in lower stem cell

activity in many in vitro studies and tumor formation in vivo (Li
et al., 2014; Tominaga et al., 2016).
Notch signaling has been demonstrated to be critical for
esophageal and breast CSCs and regulates genes that establish stemness (Cheng et al., 2016; Forghanifard et al., 2015).
It appears that Notch signaling drives resistance to chemotherapy by maintaining a robust population of CSCs (Islam
et al., 2015; Wang et al., 2014). It has been demonstrated that
Notch activity in patients biopsy samples can predict outcome to chemotherapy. Consequently, Notch/Jagged 1 signaling pathway inhibition is one of the targets in cancer
treatment. Targeting of Notch signaling in cancer using small
molecules and neutralizing antibodies against Notch has been
attempted with little success due to signaling pathway complexities and also toxicity caused by complete pathway inhibition (Li et al., 2014; Purow, 2012; Takebe et al., 2014).
Notch ligands, known for their low rate of mutations in

FIG. 4. Selected CSC-associated signaling pathways.

Several signaling pathways involved in normal cellular
processes such as the Wnt/b-catenin, NF-kb, PI3K/Akt, and
Notch/Jagged pathways are also involved in maintaining the
CSC status. Dysregulation of these pathways is usually a
requirement in maintaining the stemness of CSCs.

CANCER STEM CELLS AND THERAPEUTIC INNOVATION

687

FIG. 5. Proposed therapeutic strategies against CSCs. Elimination of CSCs can be achieved through the destruction of the
CSC tumor niche, thereby making CSC growth impossible, for example, through surface marker inhibition or blockage
using either monoclonal or small molecules, targeting cellular signaling pathways associated with CSC self-renewal and
differentiation, and last, targeting ABC transporters that are associated with drug resistance. ABC, ATP-binding cassette.
cancers and well-studied functions, are attractive targets for
cancer therapy (Li et al., 2014). Targeting Notch ligands such
as Jagged 1 offers the opportunity to selectively block specific elements of the pathway important only to tumor biology (Li et al., 2014; Purow, 2012).
The use of targeted therapy in combination with a typical
chemotherapeutic treatment strategy is the ultimate answer to
providing a more durable cure for many cancers, including liver, ovarian, and esophageal cancer. Jagged1 is one of the Notch
ligands shown to be overexpressed in many cancer types, including neck, ovarian, and esophageal cancer (Li et al., 2014;
Purow, 2012; Scott et al., 2012). The expression of Jagged1 can
be induced by several other pathways important in cancers such
as Wnt/b-catenin, NF-kb, and TGF-b (Li et al., 2014; Scott
et al., 2012). Jagged1 is directly implicated in the promotion of
CSC self-renewal in breast cancer and drives Notch signaling in
stem cells (Li et al., 2014; Purow, 2012; Scott et al., 2012).
Many reports have indicated Jagged1 as an important
player in deciding the fate of stem cells in different cancers
and thus show that tumor Jagged1 expression is a relevant
target for CSCs (Li et al., 2014; Purow, 2012; Scott et al.,
2012). Jagged1 is also implicated in the inhibition of apoptosis, metastasis, and therapy resistance in many cancers (Li
et al., 2014; Purow, 2012). CSCs have also been shown to
have increased invasive potential and are thought to be largely responsible for cancer metastasis (Kim et al., 2005,
2016; Prince et al., 2007; Schatton et al., 2008; Szotek et al.,
2006). Thus, CSCs are an emerging target for cancer therapy
and any therapy targeting CSCs holds great potential for
improving cancer treatment and outcome (Fig. 5). Studies

done using CSCs in vitro, however, do not reflect the in vivo

tumor cell behavior.
Conclusions and Future Directions

Recent data illustrate that CSCs and clonal evolution have

some degree of convergence (Anderson et al., 2011; Kreso
and Dick, 2014; Visvader and Lindeman, 2012). The CSC
phenotype therefore can be induced and altered by the microenvironment through growth factors and cytokines. Thus,
the identification and isolation of CSCs using specific
markers remain a challenge. No longer should we take it for
granted that CSC markers discovered earlier are universally
able to distinguish tumorigenic and nontumorigenic cells.
Given the heterogeneity observed in many patients, it is advisable that CSCs need to be confirmed in functional assays
in each patient or under each experimental circumstance.
Promising anti-CSC therapies so far have only reduced
rather than eradicated solid tumors in preclinical models. In
addition, the efficacy of these anti-CSC therapies is still debatable. The evaluation of CSC response to anti-CSC-directed
therapy under clinical setting trials is still to be obtained. In
conclusion, since the CSC phenotype is diverse, this has to be
taken into account when considering chemotherapeutic interventions. Any chemotherapy against CSCs will still require the
use of conventional therapy for it to be successful.
Acknowledgments

This work was supported by the International Centre for

Genetic Engineering and Biotechnology (ICGEB), the South

688

African Medical Research Council, the National Research

Foundation (NRF) of South Africa, and the University of
Cape Town. The funders had no role in the conduct of the
research or preparation of the manuscript.
Author Disclosure Statement

The authors declare that no conflicting financial interests

exist.
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Address correspondence to:

Dr. Kevin Dzobo
International Centre for Genetic Engineering and Biotechnology (ICGEB)
Cape Town Component
Wernher and Beit Building (South)
Anzio Road, Observatory
Cape Town 7925
South Africa
E-mail: kd.dzobo@uct.ac.za
Abbreviations Used
ABC ATP-binding cassette
ALDH aldehyde dehydrogenase
Bcl-2 B cell lymphoma 2
Bcl-xL B cell lymphoma-extra large
BCRP breast cancer resistance protein
BRCA1 Breast cancer type 1 susceptibility protein
CD cluster of differentiation
CSC cancer stem cell
DNA deoxyribonucleic acid
EMT epithelialmesenchymal transition
EpCAM epithelial cell adhesion molecule
FACS fluorescence-activated cell sorting
IL interleukin
JAK/STAT Janus kinase/signal transducers and
activators of transcription
MACS magnetic-activated cell sorting
MSC mesenchymal stem cell
NF-kb nuclear factor-kappa beta
PTEN phosphatase and tensin homolog
SSEA-1 antistage-specific embryonic antigen-1
TGF-b transforming growth factor beta
TIC tumor-initiating cell
TNF-a tumor necrosis factor alpha
Wnt wingless-type MMTV integration site family
member