Int. J. Mol. Sci. 2014 , 15, 8169-8185 doi:10.3390ijms15058169 [626353]

Int. J. Mol. Sci. 2014 , 15, 8169-8185; doi:10.3390/ijms15058169

International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Review
Cancer Stem Cells: Biological Functions and
Therapeutically Targeting
Marius Eugen Ciurea 1,†, Ada Maria Georgescu 1,†, Stefana Oana Purcaru 1,
Stefan-Alexandru Artene 1, Ghazaleh Hooshyar Emami 1, Mihai Virgil Boldeanu 2,
Daniela Elise Tache 1 and Anica Dricu 1,*
1 Faculty of Medicine, University of Medicine and Pharmacy of Craiova, Str. Petru Rares nr. 2-4,
Craiova 710204, Romania; E-Mails: [anonimizat] (M.E.C.);
[anonimizat] (A.M.G.); [anonimizat] (S .O.P.); stefan.artene@ya hoo.com (S.-A.A.);
[anonimizat] (G.H.E.) ; [anonimizat] (D.E.T.)
2 Stem Cell Bank Unit, Medico Science SRL, Str. Brazda lui Novac nr. 1B, Craiova 200690,
Romania; E-Mail: [anonimizat]
† These authors contributed equally to this work.
* Author to whom correspondence should be a ddressed; E-Mail: anic [anonimizat] or
[anonimizat]; Tel. : +40-0351-443-500; Fax: +40-0251-593-077.
Received: 23 March 2014; in revised form : 20 April 2014 / Accepted: 24 April 2014 /
Published: 9 May 2014
Abstract: Almost all tumors are composed of a heterogeneous cell population, making
them difficult to treat. A small cancer stem ce ll population with a low proliferation rate and
a high tumorigenic potential is t hought to be responsi ble for cancer devel opment, metastasis
and resistance to therapy. Stem ce lls were reported to be involved in both normal development
and carcinogenesis, some molecular mechanisms being common in both processes. No less
controversial, stem cells are c onsidered to be important in tr eatment of malignant diseases
both as targets and drug carriers. The efforts to understand the role of different signalling
in cancer stem cells re quires in depth knowledge about th e mechanisms that control their
self-renewal, differentiation and malignant potential. The aim of this paper is to discuss insights into cancer stem cells historical background and to pr ovide a brief review of the
new therapeutic strategies fo r targeting cancer stem cells.
Keywords: cancer stem cell; tumorigenicity; signalli ng pathways; cancer stem cell markers;
cancer therapy
OPEN ACCESS

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1. Introduction
According to the modern theory of carcinogenes is, malignant transformation may occur due to
the action of a wide range of mutageni c agents on stem cells pr esent in the adult tissue [1]. The exact origin
of cancer stem cells (CSCs) remains unknown, despite intensive research in the last decade. In a
study on leukemia, published by Bonnet et al. [2] in Nature Medicine in 1997, the existence of a
heterogeneous tumor cell population was first mentione d; this cell population was analyzed in terms of
proliferation and differentiat ion. These cells, found in leukemia cell popu lations, were though t to have stem
cells properties, such as self-renewal capacity and hi gh proliferation rate [3]. Another study conducted
by Passegué et al. [4] demonstrated that in leukemia, the pr esence of stem cells is necessary and
sufficient for maintaining the tumor cell population. It ha s also been suggeste d that the unlimited
self-renewal capacity of CSCs may be the cause of tumor recurrence [5]. It has r ecently been
demonstrated that CSCs are present in both hematologic malignancies and solid tumors ( i.e., breast
cancer, brain tumors, malignant melanoma or prosta te cancer) [6,7]. Additionally, surface markers of
the CSCs have been identified in many type s of cancers includin g: leukemia CD34+/CD38 −, breast
cancer CD44+/ESA+/CD24 −, brain cancer CD133+, multiple myeloma CD138 −, pancreatic cancer
CD44+/CD24+/ESA+, colon cancer CD133+, liver cancer CD133+ [5], prostate can cer CD44+/CD133+ [6],
lung cancer CD133+ and ovarian cancer CD133+/CD44+ /CD117+ [7]. After num erous preclinical and
clinical studies, it has been show n that adult stem cells could turn into CSCs with specific surface
markers [8,9]. Specific targeting of tumor stem cel ls has been suggested to be a good alternative for
cancer treatment [10,11]. Some studi es demonstrate that CSCs exist in primary human sarcoma tumors
such as bone sarcomas [12], and CD133 has been show n to be a potential mark er for identification of
the CSCs as seen in a paper by Suva et al. [13] where a population of Ewing’s sarcoma family tumor
(ESFT) cells expressed CD133 which also fulfilled in vivo criteria of CSCs and in vitro plasticity
properties of mesenchym al stem cells [12,13].
2. Tumor Cells vs. Tumor Stem Cells
Over the years, a variety of polemical concepts have been generated to explain the process of
carcinogenesis. In the early 1900s, scientists first beli eved that cancer is a somatic cell disorder [14]
and soon after Tyzzer, E. introduced the notion of “s omatic mutation” in connection with cancer [15].
However, Boveri’s observation [14] was crucial in understanding the pr ocess of carcinoge ns. He believed
that chromosomal abnormalities are fu ndamental to cancer development, anticipating the cancer genetic
hypothesis [14]. More convincing arguments and ev idence to sustain the cancer genetic hypothesis
came from the discovery that chemicals and radi ations could act as mutagenic factors [16,17].
The cancer genetic hypothesis was further supporte d by Knudson’s two-hit th eory, postulating that
at least two genetic mutations in a tumor suppressor gene are necessary to gene rate cancer [18]. Two-hit
hypothesis of carcinogenesis may expl ain why people with a family hist ory of cancer do not necessarily
develop malignancies. These individuals may inherit a mutated gene, but at le ast a second mutation is
needed for occurrence of cancer. This theory may also explain why pe ople with no family history of
cancer can develop cancer, as long as there are at least two geneti c mutations that may occur for a
variety of reasons [19,20]. In suppo rt of the two mutation theory, ot her clinical observations showed

Int. J. Mol. Sci. 2014 , 15 8171

that somatic mutations in the retinoblastoma gene were present in patients with several types of cancer
(e.g., sarcomas breast cancer, bladder cancer, lung cancer) [21,22].
In 1976, Nowell, P.C. proposed the multistep geneti c model of tumorigenesis [23] and in 2000,
Hanahan and Weinberg explained th e classical model of mo lecular transformation in cancer cells [24].
These studies defined the model of carcinogenesis known as the “somatic mu tation theory”, stating
that cancer is a clonal, cell-based disease, assuming that quiescence is the regular state of cells in
the body [24,25].
The “somatic mutation theory” has dominated oncol ogy for more than 40 years; it explains that
multistep genetic alteration of recessively acti ng tumor suppressor genes and dominantly acting
oncogenes take place in cells of origin, giving rise to tumor prol iferation, invasion, metastasis and
drug resistance.
However, the cellular origin of cancer and the mechanisms behind cancer development are still
debatable since tumors, be they solid or liquid, are heterogeneous cell populati ons composed of a large
number of tumor and non-tumor cell populations. From this perspective, a new model—the tissue
organization field model—tries to explain the development of cancer, meaning that cancer is a
tissue-based disease and involves a dynamic comm unication between the various cell populations
coexisting in cancer tissue and also stroma/epith elium interactions [26,27]. These models tried to
define the model of carcinogenesis, responsible for both clonal selection and tumor cell heterogeneity.
Recently, in a study by Feinberg et al. the epigenetic aspect was added to this theory which
accounts for the alterations in global DNA methyl ation that in turn can induce both abnormal
activation of proliferation genes and tumor suppresso r genes silencing [28]. In addition, the author
suggested that “tumor-progenitor genes” promote epigenetic disrupt ion of stem/progenitor cells and
that the epigenetic plasticity togeth er with genetic injuries are responsible for tumor cell heterogeneity
and tumor progression [28].
Most of current understanding ab out the existence of stem/proge nitor cells in adult tissue
originates from animal model studies [29–31], many authors suggesting th at the existence of
stem/progenitor cells and the committed progenitors or transit-amplifying cells may enable the
malignant transformation [32].
The CSCs theory is an old idea that was first described in 1973 by Moore et al. [33]. According to
the current CSCs accepted hypothesis, besides the cells that form the tumor bulk, the malignant
transformation also involves the existence of a cell population with special properties such as
self-healing ability, called cancer stem cells or tumor stem cells [34–36].
CSCs are able to self-heal and sustain tumor growth and heterogeneity. In this context, the CSCs
theory has many similarities to the evolution model of whole body. Stem cells that pass through
embryogenesis can be normal adult stem cells (ti ssue stem cell) or diffe rentiated cells. Due to
similarities with the evolution model of whole body, it is speculated that an adult stem cell that
acquires a genetic mutation, develops into a CSC of origin (CSCO) that acquires several new genetic
mutations, developing into a CSC and fi nally giving rise to cancer [37].
The CSC hypothesis gained credibility because all main cancer-origin theories (genetic/epigenetic
events, chemical-, infection-, viru s-induced carcinogenesis) indicated that the tissue stem cell is
involved in the genera tion of cancer [2,38–43].

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Unlike the traditional theory of carcinogenesis, r ecent results obtained from different research
groups suggest that CSCs are the dr iving force of tumorigenesis and metastases, and are the cause of
tumor properties, such as proliferation, aggr essiveness and resistan ce to treatment [44].
To explain how cancer occurs and progresses from CSCs, the hierarchy model, also known as clonogenic
model, has been proposed. According to the CSC clona l model, only a subset of cancer cells called
CSCs are able to initiate mali gnant progression resulting in heter ogeneous tumors (Figure 1) [45–47].
Figure 1. Theory of ma lignant transformations from adult stem cells. On the left, the stochastic
model postulates that several cancer cell type s have an equal ability to regenerate and
proliferate, each one being capab le of giving birth to another tumor (the green, pink, blue
and red cells represent similar, non-stem tumoral cell populations); On the right,
the hierarchy model postulates that only a specific population (cancer stem cells
represented by the red cells marked with a CSC in the middle) has the distinct ability to
regenerate, multiply and to differentiate into other subset populations (the green, blue and
pink cells), hence giving birth to other tumors.

In a study by Chaffer et al. , the authors demonstrated that both normal and CSC-like cells can
materialize de novo from more differentiated cell types. This discovery brings an addition to the
hierarchical models that do not take into consideration a special type of cell plasticity in which stem
and non-stem states are converted into one another, often as the result of the cell environment [48].
Consequently, researchers have us ed the stochastic model to illustrate the CSC model of cancer
development, whereby particular circumstances in a mix-tumor cell populat ion transform any tumor
cell into a stochastically tumor-i nitiating cell, leading to tumor heterogeneity (Figure 1) [44,49–51].
Tissue organization architecture model and external stimuli were suggested to be important in CSC
initiating tumors. In the paper of Vermeulen et al. , the authors demonstrated that in colon CSCs,
Wnt activity and cancer stem ness may be regulated by environmenta l stimuli. They also showed that
by reprogramming, clonogenic differentia ted cancer cells can no longer turn into CSCs, and retrieve
their tumorigenic capacity when stimulated with my ofibroblast-derived factors, suggesting that cancer
stemness is not an unyielding characteristic [52].

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Roesch et al. detected a slowly dividing cell population and demonstr ated that it could sustain
melanoma growth and self-renew. They also show ed that these cells can shift cell state through
epigenetic changes mediated by JARI D1B, implying cell plasticity [53].
The cancer associated fibroblasts (CAFs) and th e epithelial-mesenchymal transition (EMT) have
also been reported as important components of the tumor microenvi ronment induced cell plasticity.
The current reports of the CAFs acknowledge that this stimulates cancer cells to express stem cell markers
like CD133 or CD44, and stimulate lo cal acidification that in turn alters the extracellular matrix
pattern, increasing cancer cell anchorage-independe nt growth potential and their tumor-repopulating
ability [54–56].
EMT is a key factor that is often activated dur ing embryogenesis but also during cancer invasion
and metastasis [57,58]. Mani et al. [59] showed that “Induction of an EMT in non-tumorigenic and
immortalized human mammary epithelial cells results in the ac quisition of mesenchymal traits and
properties associated with mammary epithelial stem cells”. These findings illu strate a direct link
between the EMT and the acqu isition of epithelial stem -cell properties [59]. Inte restingly, the mechanisms
that promote the CAFs and EMT reactivity in cancer ce lls have been shown to be analogous, both
enrolling redox related molecules such as hypoxi a inducible factor 1 (H IF) and cyclooxygenase 2
(COX2) [60]. In addition, CAFs were shown to induce EMT by an epigenetic mechanism, which in
turn disrupts cancer cells adhesive cell-to-cel l interactions, acquiring a mesenchymal motility and
escaping from primary neoplastic lesions and metastasis [54,61].
The proposal that CSC are involved in carcinogenesis is consistent with the identification of CSC
subpopulation in leukemia [3,4,62], breast canc er [63], prostate can cer [8], ovarian ca ncer [7] and more
recently, in certain types of brain tumors [64].
The percentage of CSCs id entified in the whole tu mor cell population was no t the same in different
tumor types [50,65] and the number of the CSCs in the tumor was reported to be correlated with
patient prognosis [66,67]. However, other groups showed that tumors of the same histological type
contain a very different percenta ge of CSCs, varying from 0.03% to approximately 100% determined
by different techniques involving antibodies against surface markers such as CD133+ or using Hoechst
dye [68–71], suggesting that the number of the CSCs is of doubtful prognostic value. Other studies
using sphere cultures and differentiation identified CD133+ CSCs in osterosarcoma-stabilized cell
line by showing that only CD133+ cells were ab le to form sarcospheres. Whereas, Aldehyde
Dehydrogenase (ALDH) assays are also used to iden tify populations of CSCs w ithin breast, colon and
lung cancer but are also rarely used fo r identifying osteos arcoma cells [12].
Because CSCs divide slowly, it has also been su ggested that the CSC population is responsible for
tumor resistance to treatment.
3. Molecular Signalling Pathways in Cancer Stem Cells
Little is known about the rela tionship between CSCs and othe r heterogeneous cell populations
within a certain tumor and even less is known about si gnalling pathways that thes e cells use in order to
coordinate their behaviour. Considered “The Holy Trin ity” of cell mole cular signalling, Notch, Hedgehog
and Wnt pathways along with the B lymphoma Mo-M LV (Moloney murine leuka emia virus) insertion

Int. J. Mol. Sci. 2014 , 15 8174

region 1 homolog polycomb ring finger oncogene know n as BMI-1 pathway, are currently the most
studied molecules [72].
The Notch pathway plays a major role in both normal and CSCs. Once act ivated by its ligands
(Delta-like and Jagged ligands), Notch receptor transl ocates to the nucleus and associates with a
DNA- bound protein, starting a cascade of transduction events in the cell [73]. In the normal stem cell
population, Notch receptor is important in cellular fundamental functions su ch as proliferation,
differentiation and apoptosis. The deregulation of Notch pathway results in abnormal proliferation,
reduced differentiation and arrested apoptosis with major implications in a variety of cancers
such as: T-cell acute lymphoblastic leukemia, melanoma, breast cancer, meningioma and lung
adenocarcinoma [74,75].
Hedgehog is a molecule responsible for a plethora of effects on the early development of different
parts of the body. The Hedgehog pathway was first di scovered by experiment al biologists in the
Drosophila Melanogaster fly [76]. Af ter further research, three differe nt homologues were discovered
in the human body, each of them having a distinct role: Sonic Hedghehog (SHH), the Desert Hedgehog
(DHH) and the Indian Hedgehog (IHH) [76]. Present in both embryo and adult stem cells, the SHH is
involved in the development of the Central Nervous System (CNS), limbs a nd axial skeleton, IHH is
involved in cartilage differentiation while DHH is strongly linked to the development of germline cells
and Schwann cells [74]. The message from the He dgehog receptors is downstream transmitted through
the messenger molecules. Some proteins, such as Fused or Costal 2, have been re ported to be part of
the Hedgehog signalling, but most of the signalling pr oteins are still unknown a nd further research is
required to find the downstream receptors pathwa y [70]. Hyperactivation of Hedgehog pathway has
been linked to several types of cancer such as basal cell carcinomas , advanced prostate cancer [71],
advanced gastric adenocarcinomas [74] and medull oblastomas [77].
Another pathway involved in growth, survival and stem cell self-renewal is the Wingless
Drosophila melanogaster segment-polarity gene and Integrase-1 vertebrate homologue (Wnt) pathway.
The members of the Wnt family consist of secr eted lipoprotein ligands. By binding to various
receptors, Wnt ligands trigger receptor intracellular signa lling, switching off the GSK-3 β-dependent
degradation pathway, which in turn enables β-catenin to accumulate in the cytosol and to translocate
the nucleus to activate transcription of the Wnt targ et genes. Thus, in the active state, Wnts bind to
receptors of the Frizzled and LRP5/6 co -receptors on the cell surface leading to β-catenin protein
stabilization and triggering dislocation of the GSK-3 β kinase from the APC/Axin/GSK-3 β-complex. In
the active Wnt signals, β-catenin interplays with CK1 and the APC/Axin/GSK-3 β-complex, activating
β-TrCP/SKP pathway that induces protein ubiquitination and proteasomal degradation.
The Wnt/ β-catenin is a well conserved pathway that regulates stem cell pluripotency during
development. Aberrant Wnt signalling underlies a wi de range of pathologies in humans, including
cancer. Regarding its influence and implication in certain malignancies, the WNT pathway was
incriminated to be linked to co lorectal cancer, medulloblastoma a nd lymphoblastic leukemia. In most
cases, the common denominator of the abnormal activ ity of this pathway is gene transcription
activation by the protein β-catenin [78,79]. Several st udies on genes activated in CSCs show that
a large network of transcription fact ors (TFs) can be activated in thes e cells that are unlikely to ever
occur in normal physiological conditi ons [80]. Among diff erent CSC TFs, the Achaete-Scute Homolog 1
(ASCL1 ) gene is considered to be an upstream regulator of the Wnt si gnalling pathway. In a paper by

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Rheinbay et al. [80], ASCL1 was shown to activate Wnt pa thway by repressing the negative regulator
Dickkopf-related Protein 1 (DKK1 ) gene in glioblastoma (GBM) CSCs. It has been recently
demonstrated that ASCL1 is crucial for GBM CSCs in vivo tumorgenicity [81].
Identified as a member of the Polycomb group, the BMI 1 pathway has been directly linked to
self-renewal and differentiation of hu man stem cells. Its most significant effects have been associated
with hematopoiesis, skeleton development and ne ural growth. BMI 1 deficient mice present an
overwhelming depletion of nervous stem cells which translated into progressive post-natal
neurological retardation and other significant defects. BMI 1 depletion also results in a significant
reduction in self-renewal capacity of the HSCs [82].
BMI 1 over-expression and amplifi cation have also been found in several types of malignancies,
most notably in hematological ones such as leukemias or mantle cell lymphomas [83] due to its direct
implication in self-renewa l of hematopoietic stem cells. Othe r studies link the BMI 1 pathway to:
gliomas [82], nasopharyngeal carcinomas [84] a nd ovarian cancer [85]. R ecently, a link between the
Hedgehog pathway and the BMI-1 gene has been found in several types of can cer, such as breast,
basal cell carcinomas, medulloblastomas and esophag eal cancers [86,87]. It has b een suggested that in
breast cancer the BMI-1 gene was present in the downstream signalling of the Hedgehog pathway and that activation of the same pathway determines an upregulation of the BMI-1 expression [88].
Cleton-Jensen et al. [89] have demonstrated that miRNAs are also implicated in emergence of
CSCs from ESFTs. They were able to present a s cenario where deregulation of miRNA in CSCs could
be the cause of expression of several TFs that help generate and sustain tumorgenicity in CSC
populations. Although miRNA repression is a key feature of malignant cells, there is little evidence of
shared miRNA profiles between different types of cancer cells, this group c onsidered that because
different transcript expre ssions can be regulated by different miR NAs, it is easy to believe that CSCs
derived from different types of tumors could survive by exploiting different miRNAs to regulate
expression of their essential TFs. An example coul d be the ESFT and breast cancer CSCs that have
been demonstrated to share the same miRNA profile for c-Myc TF regulation [90].
4. Targeted Therapy against Cancer Stem Cells (CSCs)
Although advances have been achieved in ca ncer therapy by using modern techniques and
therapeutic approaches such as personalized targeted therapy, the solid tumor cells are generally
resistant to treatment and patients continue to e xhibit a poor prognosis [91]. The concept that tumor
development and progression depends on the evolution of CSCs dramatically changes the possibilities
in which cancer can be cured. This CSC populati on is very important in the tumor’s malignant
potential and response to therapy [2]. It is known th at cancer therapy targeti ng the tumor cell bulk can
produce a partial regression of the tumor, followed by the appearance of new tumor clones, developed
from existing CSC population. Ther efore, identification and target ing of neoplastic stem cells
represents a major challenge in modern cancer therapy and researchers are trying to find new
molecular therapies directed spec ifically against these cells (Fi gure 2). Thus, molecular therapies
against CSCs were reported to be more effective, as they induc e tumor regression by reducing the
occurrence of new cancer cells (Figur e 2) [49,65].

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Many studies in clinically relevant cancer therap y are based on the hypothesis that there is a strong
relationship between the tumor resistance to conventional therapy and the CSCs intrinsic mechanisms
of resistance to conventional ch emotherapeutic drugs and radiatio n therapy. Targeting CSCs or
specific pathways responsible for radiation or drug re sistance were reported to improve the treatment
effect. CSCs harbor numerous intrinsic mechanisms of resistance to conve ntional chemotherapeutic
drugs, radiation therapy, and novel tumor-targeting drugs that permit CS C survival of current cancer
therapies and CSC-mediated initiation of tumor recurrence an d metastasis [92–96].
In the last years, some insight regarding the intr acellular and extracellular mechanisms that regulate
stem cell division and differentiation has been provide d. Different intrinsic and extrinsic factors were
proposed to influence CSC expansion. These factors, present in CSCs, have been divided into the
following categories: survival, differentiation, multid rug resistance, signal-transduction and oxidative
stress factors [97]. New generatio n of cancer therapeutic drugs are designed to eliminate CSCs by
interfering with the pathwa ys mentioned above. These drugs have been examined in vivo and in vitro .
Figure 2. Targeted therapy against cancer stem ce lls. Therapeutics aimed at the general
population of cancer cells has a limited impact on cancer stem cells (CSCs). In time, their
regenerative and proliferative capabilities are respons ible for the relapse. On the contrary,
therapy aimed at the CSC population determines a destruction of the population responsible
for differentiation and proliferation which in turn produces a much more pronounced and
stable tumoral regression. The CSCs are disp layed as the green, larger cells while the
non-stem cells are depicted as the smaller, red with the skull and bones in the middle.

It has been speculated that some stem-cell signal pathways such as Wnt, Fibroblast Growth Factor
(FGF), Notch, He dgehog, and TGF β/BMP (Bone Morphogenetic Protein) signaling play an important
role in the pluripotent stem cells and CSCs homeostasis [98]. Using high throughput screening
methods, several pharmaceutical companies developed drugs that specifically target these pathways.
Inhibition of Wnt/ β-catenin signalling pathway by siRNA or by small molecule XAV939 was reported
to induce cell death in se veral types of cancer cells [99,100]. Sonic Hedgehog si gnalling has also been

Int. J. Mol. Sci. 2014 , 15 8177

evaluated as targeted pathway in regulating CSC gr owth in many cancer types. Some small-molecule
modulators of hedgehog signaling have shown su ccess in treating medulloblastoma, basal cell
carcinoma, pancreatic cancer and prostate cancer [101]. In addition, blocking the He dgehog signalling by
siRNA chemosensitized the hepato cellular carcinoma cells to 5-fluor ouracil (5-FU) treatment [102].
It is known that CSCs can be id entified by several cell surface an tigens such as CD133, CD90, CD44,
OV6, and CD326 (EpCAM). EpCAM is considered an epithelial differentiation marker, however only
when associated with stemness markers such as CD133, it can be used as a progenitor CSC marker
in experiments.
Studies show that hepatic CSCs can be iden tified using several markers including CD133, CD24,
CD44, CD90 and EpCAM [103]. A study by Chen et al. [104] demonstrated that CD133+, EpCAM+
cells, showed similar proper ties as tumor initiatin g cells (TICs) in Huh-7 cells, such as high differentiation
capacity, increased potential of colony-formation, preferential expr ession of stem cell-related genes,
MDR to some chemotherapeutics, more spheroid form ation in cell cultures and higher tumorigenicity
in NOD/SCID mice. Another study on pancreatic can cer stem-like cells demons trated that isolated
CD44+, CD133+, EpCAM+ cells of human pancreatic cancer behave as can cer stem-like cells,
which show more aggressive behavi or such as increased cell grow th and migration, clonogenicity,
and self-renewal capacity [105].
Thus, antibody-based therapeutic approaches targeting CD133, CD90, CD44, OV6, and CD326 are
currently being developed [106,107].
Although a specific antibody may be e ffective in eradicatin g CSC, it is important to note that in
most situations, this th erapeutic method is not e nough to eradicate the whol e tumor. It has been
hypothesized that tumor cells can es cape the cytotoxic effect of speci fic antibodies by decreasing the
expression of surface antige n, by developing resistance to cancer chemotherapeutic agents or by acquiring
multiple mutations. For this reason, therapeutic a pproaches targeting CSCs in combination with
traditional cancer therapies have been also used in preclinical experiments and in clinical trials [107].
Retinoic acid, a stem cell differentiation agent, has been used in combination chemotherapy in treatment
of acute promyelocytic leukemia [1 08]. Inactivation of intracellular stem cell signalling pathways in
combination with self-renewal of CSCs has also been experimented in vivo and in vitro [109,110].
Hermann and colleagues reported that a combined regime of a new hedgehog pathway inhibitor
SIBI-C1 with Rapamycin and Gemcitabine yielded very promising results. The triple treatment
completely depleted the CSCs population in primary human pancreatic cancer tissue xenografts having
a profound effect on the tumoral stro ma as well. In order to assess the effect on these therapeutic
agents on normal stem cells, blood levels were followed throughout the study and compared to a
standard gemcitabine regime. The triple combination treatment did not yield any significantly elevated
side effects as it was initi ally expected to do [111].
In another study, Guofang Chen et al. demonstrated that administration of high levels of metformin in
thyroid carcinomas inhibits growth of both thyroid ca rcinoma cells and CSCs. This effect was suggested to
be related to Metformin’s inhi bitory effect on the insulin/IGF to 5' Adenosine monophosphate
(AMP)-activated protein kinase (AMPK) and mTOR si gnalling pathways, which wa s present in both types
of cells. Due to the over-expressed nature of the insulin/IGF and AMPK/mTOR receptors in both stand and CSCs as opposed to norma l cells, the inhibitory effects were substantially different in the
two populations [112].

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A study performed by Chia-Hsin Chan et al. has shown that inhibition of the Skp2 protein, a ligase
responsible for Akt-mediat ed aerobic glycolysis, re sulted in a reduced self-renewal capacity of CSCs
in prostate cancer. Inhibition of S kp2 also increased tumor cell sensit ivity to chemotherapeutic agents.
The therapeutic agent had shown a si gnificantly higher affinity and cons equential destructive effect for
cancer cells in comparison to normal cells, where it showed a noticeably sm aller effect over the
viability of nor mal cells [113].
Even though chemothera py is the treatment of choice against most types of can cers, its effectiveness is
limited due to multi-drug resistance (MDR) devel oped by CSCs. There are three major mechanisms
proposed in order to explain MDR, but the most investigated it is th e efflux of cellular cytotoxic drugs,
related to the over-expression of multifunctional e fflux transporters, the ATP-binding cassette (ABC)
proteins. ABC efflux pumps confer protection to CSCs, shielding them from the adverse effects of
chemotherapeutic insult [90,114–116].
Two models have been proposed to explain MDR in CSCs; one sugg ests that after chemotherapy
only ABC expressing CSCs ar e able to repopulate the tumor with newly formed CSCs or differentiated
progenitor cells. The second model proposes that only CSCs that acq uire drug resistance survive under
the pressure of mutations and give rise to new and more aggressive drug-resistant ce ll phenotypes [114].
Therefore, chemoresistant CSCs ar e considered to be the Achilles’ heel of cancer [117]. Some authors
have identified two compounds: DECA-14 and rapamyci n that selectively target neuroblastoma (NB)
CSC, while having little effect on normal st em cells, preventing NB CSC self-renewal both in vitro
and in vivo [5]. However, ongoing research is focusing on the efficacy of targeting multiple key TF
that are responsible fo r CSCs’ stemness [118].
Unfortunately, most of these new therapeutic appr oaches are not highly specific for CSCs, because
the difference between the types of cell populations ex isting in the tumor is st ill difficult to find, since
several signaling pathways are invo lved in regulation of cell prolif eration of both CSCs and normal
adult stem cells. This means that th e percentage of abnorma lities including certain distinctive features
such as over expression of specific genes and/or receptors is still re latively small. Also, due to the
nouvelle nature of therapie s targeting CSC signalli ng pathways, many of the studies are still in
preclinical phases of trials where the implications of above mentioned treatments on normal stem cells
are difficult to assess. In addition, cancers can be triggered by multiple oncoge nic mutations or CSC can
suffer multiple mutations. For this reason, CSCs targ eted therapy may be limited or may even fail and
further research studies are needed to find better methods to specifically e liminate the CSC population.
5. Conclusions
Although most questions regarding the origin and certain features of CS Cs remain unanswered,
their existence within tumors is widely accepted. Al so, more clinical and expe rimental data show that
curative cancer therapy is effective only when CS C is completely eradicated. Current therapeutic
approaches against cancer have been reported to c ontrol cell proliferation a nd tumor growth but they
are not able to completely eradicate the tumor cell mass. Remaining cells often metastasize or recur in
the same location. Chemo- and radio-therapy on th e tumor bulk may lead to a better therapeutic
response, but cannot provide a long-term complete re mission of tumors. The role of CSCs in diagnosis

Int. J. Mol. Sci. 2014 , 15 8179

and therapy of cancer has recently been the subj ect of intense research. Therefore, improving
anti-cancer treatment response requires mo re accurate identification of the CSCs.
Acknowledgments
The authors acknowledge support by UE FISCDI Romania (Grant 134/2011).
Conflicts of Interest
The authors declare no conflict of interest.
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