Cancer stem cells in glioblastoma—molecular signaling and therapeutic targeting
Cancer stem cells in glioblastoma - molecular signaling and therapeutic targeting
Zhi Huang 0
Lin Cheng 0
Olga A. Guryanova 0
Qiulian Wu 0
Shideng Bao ✉ 0
0 Department of Stem Cell Biology and Regenerative Medicine, Lerner Research Institute, Cleveland Clinic , Cleveland, OH 44195 , USA
cancer stem cell; glioblastoma; therapeutic resistance; molecular targeting; tumor angiogenesis; hypoxia response; stem cell niche
Glioblastomas (GBMs) are highly lethal primary brain
tumors. Despite current therapeutic advances in other
solid cancers, the treatment of these malignant gliomas
remains essentially palliative. GBMs are extremely
resistant to conventional radiation and chemotherapies.
We and others have demonstrated that a highly
tumorigenic subpopulation of cancer cells called GBM stem
cells (GSCs) promotes therapeutic resistance. We also
found that GSCs stimulate tumor angiogenesis by
expressing elevated levels of VEGF and contribute to
tumor growth, which has been translated into a useful
therapeutic strategy in the treatment of recurrent or
progressive GBMs. Furthermore, stem cell-like cancer
cells (cancer stem cells) have been shown to promote
metastasis. Although GBMs rarely metastasize beyond
the central nervous system, these highly infiltrative
cancers often invade into normal brain tissues
preventing surgical resection, and GSCs display an aggressive
invasive phenotype. These studies suggest that targeting
GSCs may effectively reduce tumor recurrence and
significantly improve GBM treatment. Recent studies
indicate that cancer stem cells share core signaling
pathways with normal somatic or embryonic stem cells,
but also display critical distinctions that provide
important clues into useful therapeutic targets. In this review,
we summarize the current understanding and advances
in glioma stem cell research, and discuss potential
targeting strategies for future development of anti-GSC
Glioblastomas (WHO grade IV gliomas) are the most
common type of malignant tumors in central nervous system
(CNS) in adults. Glioblastoma (GBMs) remains one of the
most fatal and least successfully treated solid tumors
et al., 2007; Wen and Kesari, 2008)
. The median survival of
GBM patients treated with multimodal therapies including
surgical resection, radiation and chemotherapy is less than 16
(Stupp et al., 2005; Furnari et al., 2007)
. This poor
prognosis for GBM patients has not improved significantly
over decades, underscoring the difficulties and challenges in
effectively detecting and treating these lethal cancers. The
fundamental problem of these malignancies is their highly
infiltrative nature and extreme resistance to conventional
treatments. Aggressive invasion of GBM cancer cells into the
normal brain tissue and spinal cord often prevents complete
removal of tumor cells through surgical resections. Invading
cancer cells appear to be particularly resistant to current
therapies and are often protected by the neurovascular niche
(Furnari et al., 2007)
. In addition, the majority of patients suffer
treatment failure within 2–3 cm of the original resection cavity,
indicating that therapeutic resistance is a common feature of
GBM tumors. Collectively, these difficulties have propelled the
reevaluation of current treatments in order to achieve
maximal efficacy with minimized toxicities or side-effects.
While chemotherapy has been used for several decades in
neuro-oncology, the oral DNA methylating agent
temozolomide (TMZ) has shown effective when used concurrently with
radiation and then as adjuvant chemotherapy such that it is
now a standard practice
(Stupp et al., 2005; Wen and Kesari,
. Several targeted therapies have been tested in trials of
malignant gliomas, but to date only bevacizumab (Avastin)
has been approved by the FDA to treat GBMs
et al., 2007a, b; Friedman et al., 2009)
. Immunotherapies and
toxin-ligand conjugates have shown promise in early clinical
trials but so far lack definitive efficacy in phase III trials. Thus,
development of more effective treatments is urgently needed,
which will require new paradigms in cancer biology and more
insight into the mechanisms underlying the cancer invasion,
therapeutic resistance and tumor recurrence in GBMs.
It has been well recognized that most solid tumors consist
of heterogeneous cancer cells, as well as vasculatures,
stromal elements and inflammatory cells
. GBM displays remarkable intratumoral
heterogeneity and cellular hierarchy not only morphologically
but also in differentiation status. Cancer cell heterogeneity
and hierarchy may be resulted from both genetic and/or
epigenetic causes. Increasing evidence from hematopoietic
malignancies and solid tumors (including brain, breast,
colorectal, head and neck cancers) has strongly supported
the concept that a subpopulation of cancer cells in each tumor
has greater potential of cancer initiation and repopulation
(Lapidot et al., 1994; Bonnet and Dick, 1997; Al-Hajj et al.,
2003; Hemmati et al., 2003; Singh et al., 2003; Galli et al.,
2004; Singh et al., 2004; Ricci-Vitiani et al., 2007; O’Brien
et al., 2007; Dalerba et al., 2007; Prince et al., 2007; Schatton
et al., 2008)
. These cells were called cancer stem cells
(CSCs) or tumor-initiating or propagating cells because they
share some critical characteristics with normal stem cells,
including the capacities for self-renewal, multi-lineage
differentiation, and maintained proliferation
(Reya et al., 2001;
Vescovi et al., 2006; Bao et al., 2006a; Rosen and Jordan,
2009; Park and rich, 2009; Heddleston et al., 2010; Frank
et al., 2010)
. Although cancer stem cell or stem cell-like
cancer cell terminology is imperfect due to its distinct
differences from normal stem cell, but it does capture the
shared characteristics with normal stem cells especially
somatic stem cells. In this review, we used “cancer stem
cell” (CSC) terminology, which does not implicate that the
cellof-origin of cancer stem cells has to be normal stem cell or
progenitor, although several recent studies have
demonstrated that normal tissue stem cells can be transformed to be
cancer stem cells
(Barker et al., 2009; Zhu et al., 2009)
Cancer stem cells in malignant gliomas that were called
glioma stem cells or GBM stem cells (GSCs) have been
identified and characterized by several groups including our
(Hemmati et al., 2003; Galli et al., 2004; Singh et al.,
2004; Bao et al., 2006a, b, 2008; Lee et al., 2006; Li et al.,
. These cells are functionally defined with self-renewal
(Fig. 1), cell differentiation in vitro (Fig. 2), and tumor
propagation in vivo (Fig. 3B). Studies from a number of
groups including ours have demonstrated that GSCs display
much greater tumorigenic potential than matched non-stem
tumor cells when xenotransplanted into the brains of
immunocompromised rodents (Fig. 3B)
(Singh et al., 2004; Bao et al.,
2006a; Lee et al., 2006; Li et al., 2009)
. We and others have
shown that GSCs have the potential to differentiate into
astrocytes, oligodendrocytes and neurons (Fig. 2)
et al., 2003; Galli et al., 2004; Singh et al., 2004; Bao et al.,
2006a; Calabrese et al., 2007)
, though GSCs commonly
display aberrant differentiation signatures with multiple
lineage markers expressed in one differentiated cell. GSCs may
be resulted from genetic and epigenetic changes in neural
stem/progenitor cells or the differentiated cells such as
astrocytes after a series of mutations or epigenetic
reprogram. Although the origin of GSCs is not clearly defined,
GSCs share similar properties with normal neural stem cells
(NSCs) that endow these cells with key traits in
carcinogenesis. These properties include enhanced potentials for
proliferation, angiogenesis, invasion and modulating immune
GSCs have been implicated in several malignant behaviors
associated with GBM tumor progression. We demonstrated
that GSCs express elevated levels of VEGF (vascular
endothelial growth factor) to enhance tumor angiogenesis
(Bao et al., 2006b)
. This finding has been confirmed by other
groups determining that SDF-1 (stromal-derived factor-1, also
known as CXCL12) is an additional pro-angiogenic ligand
expressed by GSCs
(Folkins et al., 2009)
. This is particularly
significant as a humanized anti-VEGF antibody called
bevacizumab (Avastin) has demonstrated a promising
efficacy against glioblastomas, and it was recently approved by
the United States FDA for the treatment of recurrent or
(Vredenburgh et al., 2007a, b; Friedman et
. Moreover, we also found that GSCs are relatively
resistant to radiation due to preferential response of the DNA
damage checkpoint and the enhanced DNA repair capacity
(Bao et al., 2006a)
, while other groups have shown relative
resistance of GSCs to chemotherapies such as
(Liu et al., 2006; Bleau et al., 2009)
. These studies may
help understand in part why those GBM patients with a
promising radiographic response universally suffer
recurrence and/or progression of their cancers. Thus, molecular
targeting of GSCs may directly improve the efficacy of current
cytotoxic therapies and anti-angiogenic therapies. In this
review, we summarize the roles of GSCs in tumor progression
and malignant behaviors of GBMs with attention to signaling
pathways and molecular regulators involved in maintaining
the GSC phenotype, and discuss molecular strategies
targeting GSCs for the development of novel therapeutics to
improve the survival of GBM patients in the future.
CANCER STEM CELLS AND THERAPEUTIC
RESISTANCE OF GLIOBLASTOMA
GBMs are highly infiltrative tumors that display extreme
resistance to conventional radiotherapy and chemotherapy,
and often recur rapidly in a local fashion despite maximal
(Furnari et al., 2007; Wen and Kesari,
. We and others have shown that GSCs contribute to
therapeutic resistance and likely are responsible for GBM
(Bao et al., 2006a; Liu et al., 2006; Rich and
Bao, 2007; Bleau et al., 2009)
. The presence of GSCs within
GBM tumor responsible for generating the entire mass of
cancer cells has important implications for understanding the
efficacy of current therapies and the resistance issue.
Although the hierarchical relationship between GSC
population and bulk tumor remains controversial (Park et al., 2010), it
is possible that GSCs contribute to tumor repopulation after
conventional therapies. Radiation is the most effective
nonsurgical therapy for GBMs, but it is palliative indicating that
GBMs contain resistant cancer cell populations. Indeed, we
demonstrated that GSCs are more resistant to radiation than
the matched non-stem glioma cells
(Bao et al., 2006a)
response to DNA damage induced by radiation or the
radiomimic drug neocarzionstatin (NCS), GSCs preferentially
activate DNA damage checkpoint response as measured by
the activating phosphorylation of several critical checkpoint
proteins (ATM, Rad17, Chk2 and Chk1)
(Bao et al., 2006a)
As a consequence of the preferential checkpoint activation,
GSCs are more efficient in repairing the damaged DNA and
more rapidly recover from the genotoxic stress than the
matched non-stem tumor cells. Thus, GSCs display more
resistance to radiation-induced apoptosis than the non-stem
cancer cells in vitro and in vivo
(Bao et al., 2006a)
a low molecular weight inhibitor (Debromohymenialdisine,
DBH) of Chk2 and Chk1 kinases abolishes the
radioresistance of GSCs, suggesting that targeting the DNA
damage checkpoint activation may sensitize GSCs to
radiotherapy and thus overcome the radioresistance of GBMs.
Although checkpoint inhibitors may be used as
radiosensitizers of GSC population, the effects of the checkpoint
inhibitors on normal stem cells and other normal cells
must also be considered, because long-term inhibition of
checkpoint activation may lead to new oncogenesis.
Recently, it has also been shown that inhibition of the Notch
signaling pathway by the γ-secretase inhibitor or Notch
shRNA renders GSCs more sensitive to radiation (Wang
et al., 2010), suggesting that Notch pathway may serve as
another potential target for reducing GBM radioresistance. In
addition, recent studies suggested that targeting SirT1
expression or HSP90 activity can also attenuate
radioresistance of GSC population
(Chang et al., 2009; Sauvageot
et al., 2009)
. It seems that multiple molecular mechanisms
regulate GSC radioresistance, perhaps with intertumoral
variation. Interestingly, decreased radiosensitivity in breast
cancer stem cells has been linked to lower levels reactive
oxygen species (ROS) (Diehn et al., 2009) or enhanced
activity of Wnt/β-catenin signaling
(Woodward et al., 2007)
Although these mechanisms have not been reported in
GSCs, they give hope that several molecular regulators
may be targeted synergistically in GSCs in order to effectively
The current standard of treatment for GBMs also includes
adjuvant chemotherapy with temozolomide (TMZ) that is a
commonly used oral methylating chemotherapeutic agent
(Stupp et al., 2005; Wen and Kesari, 2008)
. TMZ achieves
significant cytotoxic effect on cancer cells mainly by
methylating the O6 position of guanine in DNA. This DNA adduct can
be removed by the repair enzyme
O6-methylguanine-DNAmethyltransferase (MGMT) that is expressed in varied levels
in GBMs. MGMT expression is likely regulated at several
levels but recent attention has focused on promoter
methylation that has been linked to greater sensitivity to TMZ
treatment due to reduced MGMT levels (Hegi, et al., 2005).
The addition of TMZ to GBM therapy has been potentially
most effective by a radiosensitization effect
(Stupp et al.,
, but TMZ is commonly used as adjuvant therapy as
well. Although therapy with TMZ may slow GBM tumor growth
and increases the proportion of patients surviving for two
years, long-term survivors are still rare due to drug resistance
and tumor recurrence. Invariable recurrence after TMZ
therapy indicates the presence of TMZ-resistant cancer
cells in GBM. It has been shown that GSCs also contribute
to the chemoresistance to TMZ
(Liu et al., 2006)
. In a
genetically engineered glioma mouse model, TMZ treatment
increased the side population (SP), a potential measure of
cancer stem cells
(Bleau et al., 2009)
. Although TMZ can
eliminate MGMT-negative GSCs
(Beier et al., 2008)
does not inhibit self-renewal of GSCs with normal levels of
(Clement et al., 2007)
. Several other potential
mechanisms underlying the drug resistance of GSCs have
also been reported. Increased expression of drug
transporters including the ABC (ATP binding cassette) transporters
that pump out chemotherapeutic agents may be one of critical
mechanisms. Some studies suggested that the expression of
ABC transporters increase in cancer stem cells
et al., 2008)
. A side population (SP) of cancer cells isolated
from tumors may represent a class of cancer stem cells with
high drug efflux capacity and thus show inherently high
resistance to chemotherapeutic agents
. SP cells express elevated levels of the ABC drug
transporters such as ABCG2 and ABCA3 in GBM cell lines
(Hirschmann-Jax et al., 2004)
, suggesting that targeting these
drug transporters may reduce chemoresistance of GSC
populations in GBM.
GLIOBLASTOMA STEM CELLS AND TUMOR
Active tumor angiogenesis is one of hallmarks of
(Plate and Risau, 1995; Furnari et al., 2007)
neovascularization plays crucial roles in providing nutrition
and oxygen and removing waste to facilitate the rapid growth
and progression of malignant gliomas. The degree of
vascularization is significantly correlated with the glioma
malignancy, tumor aggressiveiness, and clinical prognosis
(Plate and Risau, 1995). Based on this background, a number
of laboratories have investigated the relationship between the
tumor vasculature and GSC population. We have determined
that GSCs formed tumors with greater vascularity than the
non-stem tumor cells
(Bao et al., 2006b)
. We demonstrated
that GSCs promote tumor angiogenesis partially through
elevated expression of the most important pro-angiogenic
factor, vascular endothelial growth factor (VEGF)
(Bao et al.,
2006b; Li et al., 2009)
. GSCs express much higher levels of
VEGF than the matched non-stem tumor cells and display
greater angiogenic potential in vitro and in vivo. Targeting
VEGF through bevacizumab specifically blocked GSC
proangiogenic effects both in vitro and in vivo. Other groups
showed that GSCs promote both tumor angiogenesis and
vasculogenesis via VEGF and SDF-1
(Folkins et al., 2009)
understand the molecular mechanisms that drive VEGF
expression in GSCs, we interrogated the role of hypoxia
and the hypoxia inducible factors (HIFs) in the GSC-mediated
angiogenesis. As expected, hypoxic condition induced VEGF
expression in both GSCs and non-stem glioma cells but the
levels were consistently higher in GSCs
(Li et al., 2009;
Heddleston et al., 2010)
. Interestingly, HIF-1α and HIF-2α
specifically controlled VEGF expression in GSCs in a
(Li et al., 2009)
. In addition, hypoxia can
promote the expansion of GSC fraction and regulate
expression of stem cell markers
(Heddleston et al., 2009;
McCord et al., 2009; Soeda et al., 2009)
. Thus, hypoxia may
enhance tumor progression and therapeutic resistance
through its promotion of a cancer stem cell phenotype and
induction of VEGF and other pro-angiogenic factors.
The powerful pro-angiogenic effect of GSCs on tumor
vascularization suggests that targeting GSCs should disrupt
tumor angiogenesis. Furthermore, recent studies
demonstrated that the relationship between GSCs and the
vasculature is complex and bi-directional. Normal neural stem cells
(NSCs) reside in perivascular locations called vascular niches
that provide essential pro-survival and maintenance cues
(Shen et al., 2008; Tavazoie et al., 2008; Mirzadeh et al.,
. In a seminal study, Gilbertson group demonstrated that
cancer stem cells in brain tumors also reside in perivascular
(Calabrese et al., 2007)
. They further showed that
endothelial cells increase survival of brain tumor stem cells
and targeting the tumor vasculature with bevacizumab
reduces the number of cancer stem cells in treated tumors.
In addition, they found that co-transplantation of endothelial
cells with GSCs accelerates tumor initiation and progression
(Calabrese et al., 2007)
. We have observed that glioma cells
expressing GSC markers, CD133, HIF2α or L1CAM (CD171),
are localized near blood vessels
(Bao et al., 2006a, 2008; Li et
(Fig. 3A). Collectively, these studies suggest that
the prevascular niche may provide a specific
microenvironment for the maintenance of GSCs. The symbiotic
relationship between GSCs and vasculatures may explain the
promising efficacy of anti-angiogenesis therapy with cediranib
(AZD2171, a VEGFR inhibitor)
or bevacizumab for GBM
patients in the clinical trials
(Vredenburgh et al., 2007a, b;
Batchelor et al., 2007; Friedman et al., 2009)
antiangiogenic treatment may actually function as an anti-GSC
therapy (Folkins et al., 2007). It is likely that anti-angiogenic
drugs might not only inhibit tumor vascularization to suppress
GBM growth, but also directly disrupt the niches for the
maintenance of GSCs, therefore weakening the “tumor roots”
or eliminating the “tumor seeds” to block further tumor
HYPOXIC RESPONSES IN GLIOBLASTOMA STEM
Hypoxia is commonly present in many types of solid tumors
including GBMs. Hypoxic condition was thought to have a
negative impact on tumor growth. However, now it has been
well recognized that hypoxia actually promotes tumor
angiogenesis, cancer invasion and therapeutic resistance
such as radioresistance in GBMs
(Jensen, 2009; Heddleston
et al., 2009)
. Moreover, recent works from our group and
others have demonstrated that hypoxic niches play critical
roles in the maintenance of cancer stem cells in tumor tissue
(Li et al., 2009; Heddleston et al., 2009; Pietras et al., 2008,
. Similarly, hypoxic niches are also involved in the
maintenance of normal stem cells. For example,
hematopoietic stem cells are maintained in hypoxic niches in bone
marrow (Parmar et al., 2007). Interestingly, hypoxia also
prevents the differentiation of neural stem cells and promotes
the maintenance of self-renewal potential of embryonic stem
(Studer et al., 2000; Marrison et al., 2000; Ezashi et
al., 2005; Santilli et al., 2010)
. In addition, restricted oxygen
concentrations have been shown to enhance the production
of induced pluripotent stem cells (iPSC)
(Yoshida et al., 2009)
GBMs frequently display areas of necrosis that occurs in
avascular and low oxygen regions. Surprisingly, necrosis
serves as a grading criterium for GBMs in clinical diagnosis.
While brain tumor stem cells have been linked to a
perivascular niche, we have found an additional enrichment
of GSCs around necrotic regions that often display hypoxic
(Li et al., 2009)
. Several groups have found that
restricted oxygen promotes a GSC phenotype
(McCord et al.,
2009; Soeda et al., 2009)
. Our group also found that restricted
oxygen conditions increase expression of GSC markers and
the indicators of self-renewal, suggesting that the state of
cancer stem cell may be plastic and that microenvironmental
conditions can promote the acquisition of a stem cell-like
(Heddleston et al., 2009, 2010)
. These studies
suggest that disrupting the microenvironment of GSCs, like
the hypoxic niches, may offer a new approach targeting GSCs
The cellular responses to hypoxia are mainly mediated
through the hypoxia inducible factors (HIFs). In response to
hypoxia, both cancer cells and normal cells undergo
significant transcription modification that leads to alterations
of cellular activities, but different cells may display varied
hypoxic responses. Recently, we demonstrated that hypoxia
responses in GSCs differ from that in non-stem cancer cells.
Hypoxia differentially induces HIF2α in GSCs but not in
nonstem GBM cancer cells, while HIF1α is induced in both GSCs
and the non-stem GBM tumor cells
(Li et al., 2009)
Furthermore, HIF2α was essential only in GSCs and was
not expressed by normal neural stem or progenitor cells,
suggesting that HIF2α may represent a specific target for
GSCs or other cancer stem cells. Under hypoxic conditions,
GSCs display a specific gene expression profile different from
that induced in the non-stem cancer cells. In addition to the
increased VEGF expression, HIF2α and several HIF2α
transcriptional targets such as Oct4, Glut1 and Serpin B9
are specifically upregulated in GSCs under hypoxia
(Li et al.,
. GSCs display high levels of HIF2α under oxygen
concentration as high as 5% that is within the physiologic
range of oxygen in the brain and most tumor tissues, whereas
HIF1α is induced only at severely hypoxic conditions ( < 1%
oxygen) in both GSCs and the non-stem tumor cells
(Li et al.,
. Functional studies through RNA interference
demonstrated that both HIF2α and HIF1α are required for GSC
proliferation in vitro and GSC tumor formation in vivo, but only
HIF1α is required for the growth of non-stem GBM cancer
cells, suggesting that GSCs use both HIF1α and HIF2α for the
hypoxia response and may be able to survive better under
stress conditions. Other studies have shown that HIF1α
functions in the hypoxia-driven expansion of GSCs (Soeda et
al., 2009). Moreover, a statistical analysis of HIFs expression
in the REMBRANDT National Cancer Institute patient
database indicated that HIF2α but not HIF1α expression
levels informed negative survival of patients. In addition,
overexpression of HIF2α actually promotes cancer stem state
(Heddleston et al., 2009)
. These data suggest that
HIF2α represents a potential target specific for GSCs, since
HIF2α is not expressed in normal neural progenitors.
However, the role HIF2α in other normal stem cells needs
to be elucidated, in order to understand whether targeting
HIF2α have any negative impact on other normal adult stem
cells. Differential hypoxic response in GSCs may provide a
new strategy to target cancer stem cells in malignant gliomas.
SIGNALING PATHWAYS IN GLIOBLASTOMA STEM
The identification of cancer stem cells and the therapeutic
targeting of these cells to improve cancer treatment have
(Zhou et al., 2009)
, but our
understanding on the molecular signaling of cancer stem cells is still
in early development. Although cancer stem cells share some
critical characteristics with normal somatic stem or progenitor
cells, cancer stem cells are clearly distinct from the normal
stem cells at genetic/epigenic and molecular signaling levels.
It is obvious that elucidation of specific signaling pathways
involved in the maintenance and functions of GSCs will be
useful to develop novel strategies to improve GBM treatment.
A number of singling pathways associated with the
maintenance of GSC phenotypes have been reported
al., 2006; Park and Rich, 2009; Zhou et al., 2009)
. Here we
discuss a few of critical signaling transduction pathways
mediated from external signals to nucleus in GSCs.
Notch proteins include four members (Notch 1–4) of
transmembrane receptors. They mediate short-range cellular
communication through interaction with ligands (Jagged-1,
-2, and Delta-like-1, -3, and-4). The Notch-mediated signaling
pathway is essential for the maintenance of somatic stem and
progenitor cells by promoting self-renewal and repressing
(Lathia et al., 2008)
. It is well known that the
activation of Notch requires sequential proteolytic cleavages
by the γ-secretase complex to release Notch intracellular
domain from membrane to nucleus
(Mizutani et al., 2007;
Lathia et al., 2008)
. The nuclear translocation of the cleaved
Notch further leads to Notch-dependent transcription. The
important roles of Notch signaling in regulating self-renewal
and determining cell fate have been well established in neural
stem or progenitor cells (Lathia et al., 2008). Notch signaling
potently promotes the proliferation of normal neural stem cell
(NSC) and is required for the maintenance of neural
progenitors both in vitro and in vivo
(Mizutani et al., 2007)
Aberrant Notch signaling has been found in a number types of
tumors including gliomas
(Purow etal., 2005; Kanamori et al.,
. The role of Notch signaling in brain tumor stem cells
was initially identified in medulloblastomas. Blockade of
Notch signaling by a γ-secretase inhibitor (GSI-18) induces
differentiation and apoptosis of stem-like cells derived from
medulloblastomas and impairs the tumorigenic potential of
these cells (Fan et al., 2006). Recently, the function of Notch
signaling has been linked to cancer stem cells in malignant
gliomas, as inhibition of Notch signaling in GSCs attenuates
the formation of neurosphere-like colonies
(Fan et al., 2010)
Furthermore, Notch overexpression in a K-ras-induced
glioblastoma mouse model increased expression of NSC
marker Nestin and induced glioma formation in the NSC-rich
subependymal zone in brain
(Shih et al., 2006)
. As mentioned
above, Notch signaling has been linked to radioresistance of
(Wang et al., 2010)
, suggesting that inhibition of Notch
signaling may not only disrupt the maintenance of GSCs but
also reduce the radioresistance of GSCs. Now γ-secretase
inhibitors are in early clinical development for brain cancers.
In addition, other regulators of Notch signaling, including
Delta/Notch-like epidermal growth factor-related receptor
(DNER) and the Notch ligand Delta-like 4 (DLL4), can also
regulate GBM tumor growth
(Li et al., 2007; Sun et al., 2009;
Jeon et al., 2008)
. Anti-DLL4 therapies have demonstrated
anti-cancer stem cell activity
(Hoey et al., 2009)
has been raised as chronic DLL4 targeting can induce
neoplasia as well
(Yan et al., 2010)
. Further, other signaling
regulators such as ID4 (inhibitor of differentiation 4) and
CXCR4 also functionally interact with Notch signaling in brain
(Jeon et al., 2008; Williams et al., 2008)
. It is important
to understand the role of these interactions in maintaining the
stem-like phenotype of GSCs.
The Sonic Hedgehog signaling is one of key regulatory
pathways critical for the maintenance of several types of adult
stem cells, including neural stem cells
(Ruiz i Altaba et al.,
. The binding of Hedgehog ligands to the PTCH
receptor activates Gli signal transducers that then translocate
into the nucleus to activate or repress transcription of its
downstream genes. Aberrant Hedgehog signaling has been
linked to the development of medulloblastomas, the common
(Goodrich et al., 1997; Vorechovský et al.,
1997; Shahi et al., 2008)
. Active Hedgehog-Gli signaling is
also associated with gliomas (Shahi et al., 2008). In fact, the
key intracellular mediator Gli was originally discovered in a
(Kinzler et al., 1987)
. Moreover, Gli activity correlates
with tumor grade in a genetically engineered mouse model
(Becher et al., 2008)
. Several groups have investigated the
role of Hedgehog-Gli signaling in GSCs and found that this
signaling pathway regulates self-renewal and tumorigenic
potential of GSCs
(Clement et al., 2007; Ehtesham et al.,
2007; Bar et al., 2007; Xu et al., 2008)
. Treatment of GSCs
with the Hedgehog inhibitor cyclopamine or Gli RNA
interference suppresses self-renewal and proliferation while
increases apoptotic cell death. Importantly, inhibition of
Hedgehog-Gli signaling enhances the efficacy of TMZ to
inhibit GSC proliferation and induce cell death. Several
studies demonstrated that the inhibition of Hedgehog
signaling pathway blocks GSC tumor growth, and the viable
neoplastic cells after the cyclopamine treatment failed
to propagate tumors in vivo. Furthermore, cyclopamine
treatment has been shown to improve the effect of radiation
on GSCs. Taken together, these studies indicated that
Hedgehog-Gli signaling pathway is critical for the GSC
maintenance and targeting this pathway with pharmacologic
inhibitors may inhibit GSC growth and improve the efficacy of
conventional therapies against GBMs. Although the toxicity of
these inhibitors on normal stem cells needs to be carefully
evaluated, recent clinical studies with the Hedgehog inhibitor
GDC-0449 have shown promising responses with acceptable
toxicity in brain tumor patients
(Rudin et al., 2009; Yauch
et al., 2009)
Receptor Tyrosine Kinases (RTKs) mediate signal
transduction of multiple oncogenic cytokines or growth factors,
including the epidermal growth factor (EGF) and basic
fibroblast growth factor (bFGF) that are used in culturing
GSCs in vitro
(Lee et al., 2006)
. Among these RTK pathways,
the EGFR-mediated growth signaling through
phosphoinositide 3-kinase (PI3K)/Akt is one of the most critical and best
characterized pathways in malignant gliomas. GBMs
frequently display EGFR amplification and/or expression of the
constitutively active variant EGFRvIII that leads to increased
EGFR-Akt signaling in GBM cancer cells
(Moscatello et al.,
1998; Choe et al., 2003)
. Overexpression of EGFRvIII in
genetically engineered models induces glioma-like tumors
(Holland et al., 1998; Ding et al., 2003)
. It is not then surprising
that EGFR activity is required for the maintenance of GSCs as
EGFR kinase inhibitors attenuates GSC proliferation and
neurosphere formation in vitro
(Soeda et al., 2008; Griffero
et al., 2009)
. A number of intracellular pathways are activated
upon EGFR activation, but prominently the PI3K-Akt axis has
been strongly linked to GSC biology
(Dreesen et al., 2007;
Eyler et al., 2008)
. It has been recently demonstrated that
GSCs are more dependent on Akt signaling than the
nonstem GBM tumor cells
(Eyler et al., 2008)
inhibition of Akt with the pharmacologic inhibitors disrupts
GSC tumorsphere formation, reduces migration and invasion,
induces apoptosis in vitro , and significantly delays
intracranial tumor formation of GSCs
(Eyler et al., 2008; Bleau et al.,
2009; Gallia et al., 2009)
. Although targeting EGFR-PI3K-Akt
signaling pathway may have specific effects on GSCs to
reduce their tumorigenic potential, the results to date in
clinical trials of EGFR inhibitors have been disappointing,
suggesting that EGFR inhibition alone is an insufficient
therapeutic paradigm and prompting greater focus on PI3K
Bone morphogenetic proteins (BMPs)/transforming growth factor-β (TGF-β)
The BMPs and TGF-β superfamily includes a large number of
proteins that regulate a wide range of cellular activities during
development and injury responses. The BMPs play crucial
roles to instruct cell fate during neural development. Based on
this background, Vescovi group performed a seminal study
and demonstrated that BMPs can activate their canonical
receptors on GSCs to induce differentiation and inhibit GBM
tumor growth in xenograft models
(Piccirillo et al., 2006)
study showed that direct implantation of BMP-bearing beads
into glioblastomas slowed tumor growth laying the foundation
for a potential therapeutic strategy. The role of BMPs in GSCs
became more nuanced after the Fine group showed that
GSCs derived from some cases of GBMs epigenetically
regulate BMP receptors to shift toward a fetal phenotype to
escape the pro-differentiation effects of BMPs
(Lee et al.,
. In contradistinction, most members of TGF-β serve as
oncogenic stimuli in GBM growth through induction of
angiogenesis, immune evasion, and invasion
(Wick et al.,
. Recent studies have added a new dimension in TGF-β
oncogenesis as autocrine and paracrine loops that function to
maintain GSCs through induction of leukemia inhibitory factor
(LIF) and the SOX family members
(Peñuelas et al., 2009;
Ikushima et al., 2009)
. TGF-β inhibitors have already entered
into clinical trial and BMPs are being considered.
The Wnt-β-catenin signaling has been well studied in several
types of cancers such as colon cancer. The canonical Wnt
cascade is one of critical regulators in embryonic stem cells
and adult stem cells. Wnt-β-catenin signaling has clearly
defined roles in both normal stem cells and cancer stem cells
(Grigoryan et al., 2008)
. In brain, the Wnt signaling pathway
regulates brain development as well as proliferation and
selfrenewal of neural stem or progenitor cells in the fetal
ventricular zone, the postnatal subventricular zone and
(McMahon et al., 1990; Thomas et al., 1990;
Lie et al., 2005; Adachi et al., 2007; Kalani et al., 2008)
alterations of Wnt signaling pathway have been linked to
(Koch et al., 2001; Yokota et al., 2002)
signaling is activated predominantly in medulloblastoma of
the classic subtype (Thompson et al., 2006). Recent studies
indicated that Wnt-β-catenin signaling may contribute to
radioresistance in cancer stem cells
(Woodward et al.,
. Whether Wnt-β-catenin signaling is associated with
GSC maintenance and radioresistance requires further
investigation, but it is possible that Wnt blockade can
effectively and specifically target cancer stem cells in
The signal transducer and activator of transcription 3 (STAT3)
is a crucial transcriptional regulator involved in a wide range of
cellular activities in the central nervous system development,
immune response, stem cell maintenance and tumorigenesis.
The link between STAT3 activation and glioma biology has
become increasingly evident in recent years
(de la Iglesia et
. Hyper-activation of STAT3 has been detected in
many types of cancers including solid tumors and
hematopoietic malignancies. The oncogenic function of STAT3
depends on its specific phosphorylation on Tyr-705 that can
be attributed to aberrant activity of various upstream kinases.
STAT3 in conjunction with C/EBPβ correlates with
mesenchymal transformation of GBMs and inversely related to patient
(Carro et al., 2010)
. Based on this background,
several groups have investigated roles of STAT3 in GSCs.
Inhibition of STAT3 with specific inhibitors or targeting STAT3
with specific shRNAs disrupts proliferation and maintenance
(Sherry et al., 2009; Cao et al., 2010)
. Moreover, the
phosphorylated active form of STAT3 on Tyr-705 and Ser-727
is present in the GSC population, and this active form of
STAT3 decreases to undetectable level after differentiation
induction of GSCs (Sherry et al., 2009). Several pathways
upstream of STAT3 are active in GSCs. It has been shown
that interleukin-6 (IL6), erythropoietin and Notch signaling can
regulate STAT3 activation. Targeting these pathways inhibits
STAT3 activation, and cell growth and self-renewal in GSCs
(Cao et al., 2010; Wang et al., 2009)
. Interestingly, STAT3
also contributes to the immune regulation by GSCs (Wei et
al., 2010). Because STAT3 is involved in many cellular
activities in a wide range of cell types including normal stem
cells, STAT3 may not be a specific target for GSCs, although
STAT3 inhibitors are undergoing clinical development.
SPECIFIC CELL SURFACE MOLECULES IN
GLIOBLASTOMA STEM CELLS
Cell surface molecules differentially expressed in GSCs and
functionally associated with the maintenance of GSCs may
be ideal markers for sorting or targeting GSC population.
Several molecules, including CD133
(Singh et al., 2003,
(Read et al., 2009; Son et al., 2009; Ward et al.,
(Ogden et al., 2008; Tchoghandjian et al., 2010)
and L1CAM (Bao et al., 2008), have been identified on cell
surface of GSCs (Fig. 1B, 3A). Although CD133 (prominin-1)
has been widely used as a marker for enriching GSC
subpopulations from GBM primary tumors or xenografts,
many normal cells such as neural stem cells or progenitors
express CD133 potentially limiting its utility as a target, and
the reliability of CD133 to discriminate GSCs is not absolute
(Beier et al., 2007)
. CD15 (stage-specific embryonic
antigen1, SSEA-1; also called Lewis X antigen) originally identified as
a surface marker of mouse embryonic stem cells
Knowles, 1978; Damjanov et al., 1982)
has recently been
used as an alternative marker to enrich GSCs from some
GBM tumors in which CD133 is not an informative maker for
(Read et al., 2009; Son et al., 2009; Ward et
. But whether CD15 can be used as a target for
GSCs is not clear because the function of CD15 in normal
stem cells and cancer stem cells remains poorly understood,
and CD15 is a carbohydrate antigen expressed by normal
neural and progenitor cells (Capela and Temple, 2002) rather
than a distinct protein target. Other surface markers such as
A2B5 have been used for the enrichment of GSC population
(Ogden et al., 2008; Tchoghandjian et al., 2009)
, but further
investigations are needed to determine whether this surface
marker can be used for targeting GSCs in GBMs.
In the search for specific functional targets that are
uniquely expressed on cell surface of GSCs, we have
identified L1CAM as a differentially expressed surface
glycoprotein that plays critical roles in the maintenance,
survival and cellular functions of GSCs
(Bao et al., 2008)
L1CAM was originally identified as a cell adhesion molecule
in the nervous system and plays critical roles during nervous
(Maness and Schachner, 2007; Schmid
and Maness, 2008)
. This protein contains a cytoplasmic tail, a
transmembrane domain and an extracellular domain that can
interact with another L1CAM molecule through homophilic
binding, or EGFR, FGFR, neuropilin-1, α5β1 and αvβ3
integrins, and a number of extracellular matrix proteins
through heterophilic interaction
(Maness and Schachner,
2007; Schmid and Maness, 2008; Raveh et al., 2009)
L1CAM mediated intra- and inter-cellular signaling plays
crucial roles in regulating cell adhesion, migration, growth,
survival, and cancer cell invasion. We found that L1CAM is
preferentially expressed in GSCs relative to the non-stem
tumor cells and neural progenitor cells (Bao et al., 2008).
Targeting L1CAM with shRNAs specifically disrupts
tumorsphere formation and growth of GSCs in vitro . Furthermore,
L1CAM knockdown in GSCs remarkably suppressed the
tumor growth and increased the survival of mice bearing
intracranial GBM xenografts
(Bao et al., 2008)
. We have
determined the molecular mechanism by which L1CAM
promotes GSC maintenance and tumor growth. L1CAM
upregulates Olig2 to suppress expression of p21WAF1/CIP1.
In addition, a number of studies showed that L1CAM is
associated with chemoresistance of ovarian and pancreatic
(Stoeck et al., 2007; Sebens Müerköster et al., 2007;
Gavert et al., 2008; Raveh et al., 2009)
. We have recently
found that differential expression of L1CAM in GSCs
contributes to therapeutic resistance. Our data indicate that
L1CAM may represent a novel target for the development of
effective anti-GSC specific therapeutics.
SPECIFIC TRANSCRIPTION FACTORS AND THE
MAINTENANCE OF GLIOBLASTOMA STEM CELLS
Since cancer stem cells in GBMs share some critical
characteristics with normal neural stem cells and embryonic
stem cells, some important stem cell transcription factors
(SCTFs) involved in regulating normal stem cells are also
required for the maintenance of a GSC phenotype. These
stem cell transcription factors such as Sox2, Oct4, Nanog,
c-Myc, Olig2 and Bmi1 are critical for maintaining the
selfrenewal, proliferation, survival, and multi-lineage
differentiation potential of GSCs. Here we discuss some of them that
have been relatively well studied in GSCs.
Sox2, Oct4 and Nanog are core components of stem cell
transcription factor network and play crucial roles in
maintaining embryonic stem cells and somatic stem cells
al., 2004; Loh et al., 2006; Tay et al., 2008; Fong et al., 2008)
They are also critical factors for cell reprogram and the
generation of inducible pluripotent stem cells (iPS)
(Takahashi and Yamanaka, 2006; Park et al., 2008)
. These SCTFs
are differentially expressed in GSC subpopulation and are
important for GSC maintenance. Knockdown of Sox2, Oct4 or
Nanog with specific shRNAs attenuated the GSC phenotype
and induced GSC differentiation. Although targeting these
transcription factors leads to differentiation of GSCs, it is a
challenge to apply these factors as molecular targets for
eliminating GSC population, because these common stem
cell transcription factors are also crucial for the maintenance
of normal stem cells, such as hematopoietic stem cells and
neural stem cells.
Olig2 is a basic helix-loop-helix (bHLH) transcription factor
that is uniquely expressed in neural stem cells or progenitors
in the central nervous system (CNS). Olig2 is highly
expressed in neural progenitors that give rise to
oligodendrocytes and several subtypes of neurons
(Lu et al., 2002)
number of studies have shown that Olig2 is widely expressed
in astrocytomas and is required for tumor initiation and growth
(Ligon et al., 2004; 2007)
, suggesting a functional link
between Olig2 expression and cancer stem cells in gliomas.
Indeed, we found that Olig2 is differentially expressed in
GSCs relative to the non-stem tumor cells isolated from
almost all cases of GBM tumor specimens obtained in our
group, which indicates that Olig2 is a common marker for
(Bao et al., 2008; Li et al., 2009)
bHLH transcription factor is required for the maintenance of
multi-lineage differentiation potential of neural progenitors
and GSCs. It has been shown that Olig2 mediates GSC
proliferation and maintenance in part through suppression of
p21WAF1/CIP1, a key regulator in cell cycle control
(Ligon et al.,
2007; Bao et al., 2008)
c-Myc, one of most well known oncoproteins, has been
extensively studied for its critical role in the proliferation of
both normal stem cells and cancer cells. c-Myc may provide a
functional link to study the relationship between “stemness”
and tumorigenicity of cancer stem cells. Recently, several
studies demonstrated that c-Myc expression is elevated in
GSCs and it is required for maintaining GSCs in vitro and their
tumorigenic potential in vivo
(Wang et al., 2008)
. Early study
showed that c-Myc expression levels correlate with tumor
grade in gliomas
(Herms et al., 1999)
overexpression of c-Myc in mouse astroglia leads to brain tumors
that resemble human malignant gliomas
(Jensen et al., 2003)
In addition, c-Myc prevents cell differentiation and promotes
self-renewal of tumor cells derived from the pten/p53 double
null mouse model
(Zheng et al., 2008a)
. Taken together,
these studies support an important role of c-Myc in GSC
maintenance. However, the widespread effects of c-Myc in
normal physiology must be considered.
Bmi1 is one of polycomb group genes that normally function
as epigenetic silencers. Bmi1 has been implicated in stem cell
fate determination in several tissues
(Molofsky et al., 2003)
This protein functions as a positive regulator of neural stem or
progenitor cells. It has been demonstrated that Bmi1 is
required for the malignant transformation of both neural stem
cells and differentiated astrocytes (Bruggeman et al., 2007).
Transformation of Bmi1 wild-type neural stem cells lead to
high grade gliomas in vivo, but transformed Bmi1-deficient
neural stem cells only give rise to less malignant type of
gliomas with fewer cells expressing stem cell markers
(Bruggeman et al., 2007)
. Furthermore, Bmi1 is frequently
overexpressed in several types of human cancers including
malignant gliomas. Recently, Bmi1 has been shown to be
highly expressed in GSCs and required for GSC self-renewal
(Abdouh et al., 2009)
. In addition, similar finding for an
essential role of Bmi1 in maintaining cancer stem cell in
hepatocellular carcinomas has been reported
(Chiba et al.,
. These studies suggest that Bmi1 may be a common
maker for cancer stem cells in several types of human
REST, the repressor element 1-silencing transcription factor
also called NRSF (neuron-restricted silencing factor), is a
master neuronal repressor that plays crucial roles in
maintaining neural stem cells by inhibiting neuronal differentiation
(Ballas et al., 2005)
. REST contains a zinc-finger domain that
recognizes a conserved RE-1 element (21–23 base pairs)
within regulatory regions (promoters) of target genes to
suppress the transcription of critical genes associated with
neuronal differentiation. Interestingly, REST can be targeted
for proteasomal degradation by the ubiquitin E3 ligase
SCFβTRCP, which promotes neural differentiation
al., 2008; Zhang et al., 2009)
. It has been shown that REST
promotes oncogenesis in medulloblastomas and
neuroblastomas that usually arise from neural progenitors
(Lietz et al.,
1998; Lawinger et al., 2000)
. REST is also highly expressed in
glioblastomas and neuroblastomas (Blom et al., 2006). We
have observed that REST is differentially expressed in GSCs
isolated from some cases of GBM tumor specimens,
suggesting that targeting REST may induce GSC
differentiation. Thus, REST is a potential target for GSCs.
REGULATION OF GLIOBLASTOMA STEM CELLS
BY MICRO RNAS
miRNAs are a group of small non-coding RNAs that potently
silence gene expression through post-transcriptional
modification on target mRNAs. Since a single miRNA may
regulate several or many distinct mRNAs, miRNAs are
powerful regulators of gene expression. The roles of miRNAs
in regulating embryonic stem cells, somatic stem cells or
cancer stem cells have received much attention in recent
years. miRNAs are emerging as crucial regulators of cellular
proliferation and differentiation. They can function as either
tumor suppressors or oncogenes in various tissues or tumors.
miRNA has been shown to be critical in the regulation of
cancer cell functions in malignant gliomas. For example,
miRNA-21 is overexpressed in GBM tumors and functional
blockade of this miRNA induces apoptotic cell death
. However, levels of miR-124, miR-137 and miR-451
are significantly reduced in malignant gliomas (both grade III
and grade IV) relative to normal brain
(Silber et al., 2008; Gal
et al., 2008)
. The roles of miRNAs in GSCs have been
demonstrated in two recent studies showing that miR-124,
miR-137 and miR-451 levels are significantly reduced in
GSCs relative to non-stem tumor cells
(Silber et al., 2008; Gal
et al., 2008)
. Moreover, overexpression of these miRNAs in
GSCs suppresses proliferation and induces differentiation of
GSCs, indicating that these miRNAs have important roles in
maintaining a GSC phenotype. Further, external expression
of miR-451 disrupts tumorsphere formation and suppresses
tumor growth of GSCs in vivo, suggesting a tumor suppressor
role of miR-451 in GBMs
(Gal et al., 2008)
. These studies
indicate that some critical miRNAs can be potentially used as
molecular targets or therapeutic agents for targeting GSCs.
However, we may face a great challenge to deliver these
miRNAs into cancer cells in tumor tissue and to make these
miRNAs as stable targeting agents for GSCs.
DIFFERENTIATION OF GLIOBLASTOMA STEM
One of important characteristics that GSCs share with normal
neural stem cells is their multi-lineage differentiation potential,
although differentiation capacity is not considered to be one of
essential property for cancer stem cells in other tumors. We
and others have demonstrated that GSCs isolated from GBM
primary tumors or xenografts have the potential to
differentiate into cells with the marker profiles and morphologies of
astrocytes, oligodendrocytes and neurons (Fig. 2)
(Bao et al.,
. These differentiated cells usually lose long-term
repopulation potential in vitro and fail to propagate tumors in
vivo, suggesting that inducing GSC differentiation may be a
practical strategy to eliminate GSC population in GBMs.
Thus, understanding signal transduction pathways controlling
stem cell and cancer stem cell differentiation is of importance.
A number of signaling regulators involved in differentiation
induction of cancer stem cells have been identified. As
mentioned above, the members of BMP family induce GSC
differentiation into astroglial and neuron-like cells and thus
inhibit GSC proliferation and deplete GSC population
(Piccirillo et al., 2006). Study from the Viscovi group has
shown that targeting GSCs with BMP4 in vivo significantly
suppressed GBM tumor growth and reduced tumor invasion
(Piccirillo et al., 2006)
. Recent study by the Fine group has
confirmed that BMPs promote glial differentiation of GSCs
(Lee et al., 2008)
, but they also observed that GSCs derived
from some GBM tumor samples displayed enhanced cell
proliferation rather than differentiation in response to BMP
treatment. This is because GSCs from these tumor samples
lost BMPR1B expression due to epigenetic silencing by an
(Lee et al., 2008)
expression of BMPR1B restored the BMP4-induced
differentiation in these GSCs. These studies suggested that
epigenetic characteristics of individual tumor may determine
GSC response to the differentiation-inducing agents, and
BMPs in combination with epigenetic modulators may be able
to enhance differentiation of GSCs.
In addition, there are other signaling pathways or
regulators that have been implicated in controlling GSC
differentiation. Recent studies demonstrated that inactivation of PTEN
(a well-known tumor suppressor) promotes undifferentiated
state of GSCs
(Zheng et al., 2008a, b)
, suggesting PTEN may
promote GSC differentiation. PTEN is a phosphatase with
dual-specificity for both protein and lipid and is often mutated
(Fan et al., 2002; Zheng et al., 2008a)
. It has been
well known that PTEN deletion or functional loss is linked to
progression and/or immunoresistance of malignant gliomas
(Parsa et al., 2007). Inactivation of PTEN leads to increased
expression of c-Myc that is critical for maintaining GSC
proliferation and self-renewal, suggesting promoting PTEN
function may suppress the “stemness” of GSCs and induce
GSC differentiation. In another study, Sox11 has been shown
to induce GSC differentiation
(Hide et al., 2009)
Overexpression of Sox11 inhibits tumorigenic potential of GSCs by
promoting neuronal differentiation. Moreover, epigenetic
silence of Sox11 expression was detected in GSCs derived
from some GBM tumors when a gene expression profile was
analyzed between tumorigenic and non-tumorigenic clones of
glioma cancer cells. These studies suggest that inducing
differentiation is an attractive strategy to target GSCs,
although the molecular mechanisms underlying the control
of GSC differentiation is not fully understood.
Functional characterization of cancer stem cells in tumor
progression and therapeutic resistance has altered our
understanding of tumor biology, which led to a reevaluation
of conventional therapies for malignant gliomas and other
cancers. Although controversy still exists as to the methods
for isolating and characterizing GSCs and defining their exact
roles in malignant behaviors in vivo, GSCs represent a
subpopulation of cancer cells with extraordinary capacities to
promote tumor angiogenesis, invasion, therapeutic
resistance and repopulation after treatment, making them a crucial
cell population that should be targeted for anti-GBM
therapies. Novel therapies directed against GSCs may
significantly improve the currently poor record of clinical
activity with conventional treatments. Cancer cure requires
elimination of both GSC and non-stem tumor cell populations.
As non-stem tumor cells may be able to reprogram into stem
cell-like cancer cells under certain conditions, we believe that
eradicating both GSC and non-GSC cancer cell populations
in GBM is essential to achieve therapeutic success (Fig. 4).
Recent advances in this exciting research area have allowed
us to gain remarkable insights into the molecular mechanisms
or signaling pathways that are differentially present or
regulated in GSCs or non-stem tumor cells. We have
discussed several key signaling pathways or molecular
targets that are potentially useful for the future development
of anti-GSC therapeutics. Most of them are still far away from
the clinical application at this point, although the anti-vascular
niche treatment has shown promising results in clinical trials
leading to FDA approval for bevacizumab for the treatment of
recurrent or progressive GBMs. Additional translational
research is needed to validate the clinical relevance of
these laboratory findings and better apply these new
concepts to clinical practice. For example, current
radiographic endpoints examine total tumor volume rather than
specific cancer cell subpopulations. But the properties of
tumor heterogeneity and cellular hierarchy within a solid
tumor indicate that the nature of the surviving cancer cells
after treatment may determine the scope of tumor recurrence
and its lethality. Cellular and molecular analysis of tumor
heterogeneity may accelerate biomarker development and
the application of personalized medical therapy. However,
great challenges lay ahead as GSC populations themselves
are also heterogeneous
(Piccirillo et al., 2009)
and the GSCs
may evolve over time within a GBM patient. As the genetics of
gliomas are becoming increasingly defined with clear
subgroups of tumors evolving from low grade to high grade
with greater malignancy, our understanding of GSC diversity
and GBM heterogeneity will certainly become more nuanced.
It is very clear that micro-environment is crucial to maintain
GSC population. GSCs interact with not only vascular niche
but also non-stem tumor cells, stromal elements and immune
cells. The emerging concepts and roles of cancer stem cells
are still rapidly evolving. Recent studies demonstrated that
the epithelial-mesenchymal transition (EMT) plays an
important role in the acquisition of malignant and stem cell traits of
(Mani et al., 2008; Radisky and LaBarge, 2008;
Gupta et al., 2009)
. It has been well known that EMT
promotes tumor invasion and metastasis
Weinberg, 2009; Thiery et al., 2009)
. Thus, the stem cell-like
phenotype may contribute to tumor invasion and metastasis.
These paradigms are exciting as they may provide new
avenues for developing novel therapeutics to improve tumor
treatment and reduce tumor metastasis and recurrence that
cause most cancer deaths. Since the origin of GSCs in GBM
from different patient may vary and they may also display
different genetic and epigenic changes in complex tumor
tissues, future treatment for GBM and other tumors may rely
on a unique combination of several targeted therapies based
on the cellular, molecular, genetic and epigenic information of
the tumor in the individual patient. It is likely that the exciting
advances in these emerging areas of cancer research may
bring new opportunities for a group of cancer patients who
lack effective treatment options.
BMPs, bone morphogenetic proteins; CSC, cancer stem cell; CNS,
central nervous system; EGF, epidermal growth factor; GBMs,
glioblastomas; GSCs, GBM stem cells; TGF-β, transforming growth
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