Therapeutic targeting of cancer stem cells.

Abstract

Recent breakthroughs in translational oncology are opening new perspectives for the treatment of cancer. The advent of targeted therapies has provided the proof-of-concept to selectively turn-off deregulated oncogenic proteins, while the identification and validation of predictive biomarkers of response has allowed to improve, at least in some cases, their performance. Moreover, a subpopulation of tumor-propagating cells has been identified from many solid and hematological tumors. These cells share functional properties of normal stem cells, and are commonly referred to as cancer stem cells (CSCs). It is emerging that CSCs are defended against broadly used anticancer agents by means of different, partly interconnected, mechanisms. However, CSCs rely on specific pathways involved in self-renewal that can be pharmacologically antagonized by experimental molecular targeted agents, some of which have recently entered early phases of clinical development. Here, we discuss the spectrum of pharmacological strategies under clinical or preclinical development for CSCs targeting.

Full text

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PersPective Article
published: 17 June 2011
doi: 10.3389/fonc.2011.00010
Therapeutic targeting of cancer stem cells
Marcello Maugeri-Saccà1, Ann Zeuner1 and Ruggero De Maria1,2*
1 Department of Hematology, Oncology and Molecular Medicine, Istituto Superiore di Sanità, Rome, Italy
2 Mediterranean Institute of Oncology, Viagrande, Catania, Italy
Recent breakthroughs in translational oncology are opening new perspectives for the treatment of
cancer. The advent of targeted therapies has provided the proof-of-concept to selectively turn-off
deregulated oncogenic proteins, while the identification and validation of predictive biomarkers
of response has allowed to improve, at least in some cases, their performance. Moreover, a
subpopulation of tumor-propagating cells has been identified from many solid and hematological
tumors. These cells share functional properties of normal stem cells, and are commonly referred
to as cancer stem cells (CSCs). It is emerging that CSCs are defended against broadly used
anticancer agents by means of different, partly interconnected, mechanisms. However, CSCs
rely on specific pathways involved in self-renewal that can be pharmacologically antagonized
by experimental molecular targeted agents, some of which have recently entered early phases
of clinical development. Here, we discuss the spectrum of pharmacological strategies under
clinical or preclinical development for CSCs targeting.
Keywords: cancer stem cells, chemoresistance, self-renewal pathways, molecular targeted agents
Edited by:
Silvia Giordano, University of Torino,
Italy
Reviewed by:
Justin Lathia, Lerner Research
Institute, USA
Saverio Minucci, European Institute of
Oncology, Italy
*Correspondence:
Ruggero De Maria, Department of
Hematology, Oncology and Molecular
Medicine, Istituto Superiore di Sanità,
Viale Regina Elena 299, 00161 Rome,
Italy.
e-mail: ruggero.demaria@iss.it
tumor-supportive ability of the microenvironment by participating
in tumor vasculogenesis through the direct differentiation into vas-
cular cells (El Hallani et al., 2010; Ricci-Vitiani et al., 2010; Wang
et al., 2010b; Soda et al., 2011). If many functional properties of
CSCs are thought to account for the limited efficacy of chemo-
therapy, the refinement of knowledge on adult stem cells and their
malignant counterparts is revealing unexpected ways for developing
innovative anticancer agents (Figure 1).
StrategieS to revert chemoreSiStance
Adult stem cells maintain tissue function throughout life. To accom-
plish this function, stem cells are protected from endogenous or
exogenous insults to avoid exhausting their replicative function. For
instance, evidence indicates that adult stem cells survive cytotoxic
injuries and then reconstitute the damaged tissue (Dekaney et al.,
2009). Growing evidence indicates that CSCs possess similar stem
cell properties that protect them against chemotherapy.
It is known that cancer cells improperly activate DNA repair path-
ways to overcome chemotherapy-induced cell death (Hoeijmakers,
2001). Key effectors of the DNA damage response machinery have
been evaluated in clinical studies to determine the benefit of cancer
patients from chemotherapy, even though the predictive value of
DNA damage repair-linked biomarkers remains to be addressed
(Vilmar and Sørensen, 2009). It has been demonstrated that glio-
blastoma stem-like cells (GBM-SCs) repair ionizing radiation-
induced DNA lesions more readily that differentiated glioma cells
through the activation of ataxia telangiectasia mutated (ATM) and
checkpoint kinase 1 (Chk1; Bao et al., 2006). Likewise, both colon
(Gallmeier et al., 2011) and lung (our unpublished data) CSCs
aberrantly exploit the ATR/Chk1 axis to escape chemotherapy
cytotoxicity, as demonstrated by the observation that Chk1 inhi-
bition sensitized CSCs to different chemotherapeutic agents induc-
ing mitotic catastrophe. Agents interfering with DNA repair have
recently entered clinical development. The molecular background
the cancer Stem cellS concept
The idea that cancer originates from stem cells traces back to the
“embryonal rest theory,” asserting that cancer arises from embry-
onic remnants persisting in adult tissues. However, the “cancer stem
cell model” has captured great interest only in recent years following
the isolation of a rare cellular fraction of leukemia-initiating cells
with stem cell-like features (Bonnet and Dick, 1997). Ever since, this
concept has been corroborated by the isolation of cancer stem-like
cells, commonly referred to as cancer stem cells (CSCs), from many
solid tumors ranging from highly prevalent cancers (Al-Hajj et al.,
2003; Ricci-Vitiani et al., 2007; Eramo et al., 2008) to less common
neoplasms such as glioblastoma multiforme (Singh et al., 2003;
Galli et al., 2004) and thyroid cancer (Todaro et al., 2010). This
new paradigm implies that oncogenesis has its epicenter in a tissue-
resident stem cell. Thus, a tumor is hierarchically organized, similar
to adult tissues, with a CSC at the top of the pyramid that serves
as a precursor of the whole population. The discovery of CSCs has
questioned the “clonal evolution model” which, tracing its roots
to the Darwinian evolutionary principle, postulated that different
mutant clones acquire a survival advantage as a consequence of the
natural competition with other clones. However, the hierarchical
and clonal evolution models are not mutually exclusive as recently
demonstrated by the genetic heterogeneity of cancer propagating
cells, which suggests a clonal evolution within the stem cell pool
(Anderson et al., 2011). It is conceivable that transformed stem cells
maintain, although in a distorted manner, stem cell traits such as
defensive ability against chemicals and mechanisms involved in
self-renewal. Consistent with this, chemotherapy-induced death
stimuli are constrained in a multifaceted way, such as through
increased DNA repair ability and high expression of multidrug
resistance (MDR) efflux pumps (Eyler and Rich, 2008). Conversely,
the pharmacological inhibition of self-renewal-related pathways
selectively depletes CSCs in different preclinical models (Bar et al.,
2007; Hoey et al., 2009). Moreover, CSCs directly contribute to the

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Maugeri-Saccà et al. Targeting cancer stem cells
breast cancer (O’Shaughnessy et al., 2011). The synthetic lethality
concept could be also exploited for developing Chk1 inhibitors.
When exposed to DNA-damaging agents, p53-deficient cells are
unable to undergo G1 arrest and rely on Chk1 to activate cell cycle
checkpoints (Zhou and Elledge, 2000). Thus, the pharmacologi-
cal abrogation of Chk1 could selectively kill cancer cells with p53
defects. Chk1 inhibitors have recently entered clinical trials com-
bined with different antiblastic compounds, although clinical data
are not yet available.
underlying the development of these compounds is a modality of
gene–gene interaction known as synthetic lethality. According with
this model, the co-occurrence of two events, the first genetic and the
second pharmacological, results in the inhibition of two redundant
pathways that finally lead to cell death. Poly-ADP ribose polymerase
(PARP) inhibitors are the DNA repair-interfering agents at the
most advanced stage of clinical development for the treatment of
breast (Tutt et al., 2010) and ovarian (Audeh et al., 2010) cancers
carrying BRCA1 or BRCA2 germline mutations and triple-negative
Figure 1 | emerging pharmacological strategies for CSCs targeting include
self-renewal pathway antagonists and chemoresistance-reverting agents.
Self-renewal-linked signals (left) such as Hedgehog, Notch, and Wnt/β-catenin
pathways can be antagonized by molecular targeted agents including ligand-
binding molecules, receptor antagonists, or agents inhibiting intracellular
effectors. Chemosensitivity (right) could be restored by inhibiting MDR efflux
pumps that extrude chemotherapeutic agents of natural origin, or by interfering
with the DNA repair machinery that removes DNA-alkylating agent adducts.
CSCs, cancer stem cells; GSKβ, glycogen synthase kinase 3β; APC,
adenomatosis polyposis coli; TCF/LEF , T-cell transcription factor/lymphoid
enhancer-binding factor; DLL4, delta-like ligand 4; SMO, smoothened; PTCH1,
patched; GLI, glioma-associated oncogene homolog; MDR, multidrug resistance.

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Maugeri-Saccà et al. Targeting cancer stem cells
known that a considerable percentage of patients with apparent
organ-confined disease will experience distant recurrence years
later radical surgery and adjuvant systemic therapy. Thus, it is
conceivable that disseminated cancer cells are able to remain
quiescent for years (“tumor dormancy”), thus making CSCs the
ideal candidate for explaining this temporal pattern of recurrence.
Hematological malignancies are representing the benchmark for
the development of anticancer agents forcing dormant cancer cells
to proceed through the cell cycle. Recent studies have revealed that
some cytokines, such as interferon-alpha and granulocyte colony-
stimulating factor, or arsenic trioxide efficiently promote cycling
of dormant leukemic stem cells, thus representing a promising way
for restoring chemosensitivity (Essers and Trumpp, 2010). Others
drugs that could be exploited for inducing exit from quiescence
are histone deacetylase inhibitors (HDACis). These compounds act
at the epigenetic level producing different effects spanning from
apoptosis to differentiation, and the first-in-class HDACi vorinostat
has been approved for treating refractory cutaneous T-cell lym-
phoma (Lane and Chabner, 2009). It has been demonstrated that,
unlike imatinib alone, HDACis combined with imatinib induce
apoptosis in quiescent chronic myelogenous leukemia stem cells
(Zhang et al., 2010). However, it is important to underline that
chemotherapy and molecular targeted agents are significantly
more active in hematological malignancies than in solid tumors.
In this latter case, pharmacological strategies for breaking tumor
dormancy should be carefully evaluated, especially when developed
in the adjuvant setting in the attempt to eradicate minimal residual
disease. Paradoxically, maintaining disseminated cancer cells in a
quiescent state may represent an alternative way for achieving long-
lasting recurrence-free interval in solid tumors, the priority goal
in the adjuvant setting.
targeting Self-renewal pathwayS
The aberrant activation of self-renewal-linked signals is thought
to be the main determinant of CSCs fate. The Hedgehog (Hh),
Notch, and Wnt/β-catenin are the most studied and character-
ized pathways.
rationale and StrategieS for targeting the hedgehog
pathway
The Hh pathway plays a crucial role during mammalian devel-
opment and becomes later silenced in adult tissues (Ingham and
McMahon, 2001). The interest on Hh in cancer biology comes from
evidences demonstrating the inappropriate reactivation of its signal-
ing in many tumors (Merchant and Matsui, 2010). In the absence of
ligand stimulation the transmembrane-spanning receptor patched
(PTCH1) maintains the pathway in the “off” state by repressing
the activity of the serpentine receptor smoothened (SMO). Upon
ligand binding, PTCH1 inhibition on SMO is relieved, allowing
SMO to engage downstream effectors consisting in glioma-asso-
ciated oncogene homolog (GLI) transcription factors. Target genes
are involved in many cellular functions such as proliferation, sur-
vival, metastatization, and pathway auto-regulation.
The tumor-enhancing activity of aberrant Hh signaling occurs
through different and tumor-specific modalities. Among these, the
mutation-driven manner is the best characterized, resulting from
the constitutive activation of the transduction machinery as the
DNA repair pathways compete with apoptotic signaling to
decide the fate of damaged cells. However, CSCs display a ten-
dency toward an anti-apoptotic state that favors cell survival fol-
lowing chemotherapy (Signore et al., 2011). For instance, it has
been demonstrated that interleukin-4 (IL-4) is associated with the
overexpression of anti-apoptotic mediators and induces a chemore-
sistant phenotype in colon CSCs (Todaro et al., 2007). Since IL-4
is overexpressed in many epithelial cancers (Todaro et al., 2008), it
is conceivable that other types of CSCs exploit IL-4 as a defensive
mechanism.
The combination of differentiation-inducing agents and chemo-
therapy can cure the majority of patients affected by acute pro-
myelocytic leukemia (Sanz et al., 2004). The use of differentiation
therapy may not be limited to leukemia. Recently, a randomized
phase II trial demonstrated an increased response rate in non-small
cell lung cancer patients when all-trans retinoic acid was associated
with platinum-containing therapy (Arrieta et al., 2010). The iden-
tification of CSCs has fostered the identification of molecules with
pro-differentiation effects. The pro-apoptotic/pro-differentiative
bone morphogenetic protein 4 (BMP4) sensitizes colon CSCs to
5-fluorouracil and oxaliplatin, and eradicates CSC-derived tumors
in animal models (Lombardo et al., 2011). Likewise, GBM-SCs
exposed to BMP4 displayed reduced clonogenic ability coupled
with an increased expression of neural differentiation markers
(Piccirillo et al., 2006).
Next, both adult stem cells and CSCs may express high levels of
MDR pumps (Moitra et al., 2011). This protective system extrudes
from cancer cells a broad range of amphiphilic compounds includ-
ing taxanes, anthracyclines, and vinca alkaloids. The ability to pump
out different chemicals is currently exploited for the HOECHST dye
efflux assay, a technique used for CSCs isolation that define them as
side population (SP). Acute myeloid leukemia SP (Wulf et al., 2001)
and neuroblastoma SP (Hirschmann-Jax et al., 2004) are character-
ized by a greater ability in extruding different chemotherapeutic
agents compared with the non-SP. Moreover, long-term exposure
of breast cancer cells to doxorubicin resulted in the acquisition of
stem-like and chemoresistant properties (Calcagno et al., 2010),
as documented by the overexpression of both self-renewal-related
and MDR-related genes. Although first and second generation ABC
inhibitors failed to demonstrate a clinical benefit, more potent third
generation antagonists have been synthesized and are undergoing
clinical development (Wu et al., 2008). However, it has been dem-
onstrated that the SP does not always enrich for stem-like cells, at
least when evaluating GBM cell lines and primary cells (Broadley
et al., 2011), confirming previous findings indicating that chem-
oresistance of GBM-SCs is independent of the activity of ABC
transporters (Eramo et al., 2006).
Quiescence physiologically defends adult stem cells against
harmful insults and avoids the exhaustion of their replicative
potential (Wilson et al., 2008). Likewise, CSCs usually exhibit a
slow proliferation kinetics as demonstrated by label-retaining
approaches, which revealed that label-retaining cells meet the
operative criteria to be defined CSCs and survive chemotherapy,
unlike the non-label-retaining population (Dembinski and Krauss,
2009; Gao et al., 2010). Since chemotherapy is active against rap-
idly dividing cells, it is conceivable that prolonged exit from cell
cycle enables CSCs to survive chemo-radiotherapy. Moreover, it is

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Maugeri-Saccà et al. Targeting cancer stem cells
cytostatic, rather than cytotoxic, effects. Thus, it is arguable that
the evaluation of parameters of rapid tumor response in proof-
of-concept studies might underestimate the benefit from this class
of drugs when evaluated in patients whose tumors display wild-
type Hh effectors. Based on this assumption, the pharmacological
inhibition of Hh could offer greater opportunities as adjuvant
therapies in order to prevent distant recurrence. An attractive
hypothesis could be that while during early phases of the natural
history of tumors (i.e., the adjuvant setting) cancer cells may rely
on a tumor-supportive microenvironment, later phases (i.e., the
metastatic setting) could be biologically characterized by the self-
sufficiency of cancer cells. Such acquired ability of cancer cells to
thrive in a microenvironment-independent manner could result
in insensitivity to molecular targeted agents acting by depriving
cancer cells of paracrine-acting stimuli. Moreover, GDC-0449
pharmacokinetics is influenced by several factors, including solu-
bility-limited absorption, slow rate of metabolic elimination and
interaction with plasma proteins. In particular, both total level
and genetic variants of the plasma protein alpha-1-acid glycopro-
tein (AAG) seem to account for the interindividual variability of
GDC-0449 bioavailability (Graham et al., 2011), thus suggesting
that alternative schedules should be investigated in clinical tri-
als. The disappointing results from the studies mentioned above
raise the need for alternative strategies of Hh inhibition. Since
Hh transduction machinery converges on the GLI transcription
factors, small-molecule inhibitors of GLI proteins (GANT61 and
GANT58; Lauth et al., 2007) or, although less specific, indirect
inhibitors of GLI activity (PI3K/AKT inhibitors) could allow a
deeper inhibition of Hh signaling. Alternatively, ligand inhibition
through neutralizing antibodies (5E1 antibody) or ligand-binding
molecules (robotnikinin) have been proposed to avoid blocking
Hh pathway in the whole spectrum of its developmental func-
tions (Yauch et al., 2008; Stanton et al., 2009), which is of utmost
importance when treating young patients. In summary, while Hh
inhibition is emerging as a new opportunity in cancer therapy,
early clinical data are conflicting and mirror the heterogeneity
of mechanisms sustaining pathway activation. Thus, the tumor-
dependent activity of Hh signaling has to be fully dissected for
optimal clinical testing of Hh pathway inhibitors.
rationale and StrategieS for targeting the notch pathway
Notch is a short-range-acting communication system that exerts its
function via cell-to-cell contact. The developmental Notch pathway
has been implicated in various pro-tumorigenic activities spanning
from cell survival to motility (Koch and Radtke, 2007). In mammals,
the pathway is composed by four transmembrane receptors (Notch
1–4) and five ligands [delta-like ligand (DLL) 1, 3, 4, Jagged 1, and
2]. The pairing of Notch ligand-receptor leads to conformational
changes in the receptor that undergoes two sequential cleavages
operated by the “A disintegrin and metallopeptidase” 10 and 17
and gamma-secretase enzymes. The resulting Notch intracellular
domain (Nic) is released and translocates to the nucleus where it
interacts with co-activators to finally generate the Notch tran-
scriptional complex. Moreover, Notch communicates with other
oncogenic signals including the NFkB pathway, the hypoxia sen-
sor HIF1α and the estrogen receptor alpha (Pannuti et al., 2010).
The four Notch paralogs are also endowed of tumor-suppressive
consequence of loss- or gain-of-function mutations in negative or
positive controllers, respectively (Xie et al., 1997, 1998). Germline
mutations in PTCH1 underpin the Gorlin syndrome (Hahn et al.,
1996), a condition associated with the predisposition to develop
multiple basal cell carcinoma. Moreover, many sporadic forms of
basal cell carcinoma are characterized by a comparable mutation
pattern (Daya-Grosjean and Couvé-Privat, 2005). Likewise, PTCH1
knockout mice revealed that aberrant Hh pathway activation causes
unrestricted proliferation of cerebellar stem cells that results in
the onset of medulloblastoma (Yang et al., 2008). Consistent with
this, genome-wide expression profiles unveiled that up to one-third
of medulloblastoma patients carry somatic mutations in SMO,
PTCH1, or downstream effectors (Thompson et al., 2006). While
activating/inactivating mutations sustain aberrant Hh activation
in a niche of tumors, a dual paracrine model is probably the main
modality of Hh activation in many cancers (Dierks et al., 2007;
Yauch et al., 2008). Accordingly, the overproduction of Hh ligands
by cancer cells activates the stromal compartment that responds
with the production of growth factors. Alternatively, ligands
secreted by stromal cells exert their activity on recipient cancer
cells. To further complicate this picture, pathway trans-activation
via the PI3K/AKT axis (Riobo et al., 2006), KRAS (Ji et al., 2007),
and TGF-β (Dennler et al., 2007) has been documented.
Increasing evidence is connecting the Hh pathway with CSCs.
The control of the self-renewal by Hh has been documented in
chronic myeloid leukemia (Zhao et al., 2009) and, through the
modulation of Bmi-1, in breast CSCs (Liu et al., 2006). In multiple
myeloma a marked asymmetry has been reported in Hh pathway
components distribution and sensitivity to pathway inhibition
between B-cell-like progenitors and mature myelomatous plasma
cells (Peacock et al., 2007). Moreover, Hh pathway inhibition sig-
nificantly hampers GBM-SCs clonogenicity (Bar et al., 2007) and
preferentially depletes pancreatic CSCs (Feldmann et al., 2007).
The first evidence suggesting that Hh signaling could be phar-
macologically antagonized came from the identification of the
teratogenic steroidal alkaloid cyclopamine (Chen et al., 2002).
Subsequently, high-throughput screening of small-molecule
libraries led to the identification of several SMO antagonists
(Frank-Kamenetsky et al., 2002). The first-in-class oral SMO
inhibitor GDC-0449 was initially tested in a phase I study enroll-
ing 33 patients with locally advanced or metastatic basal cell
carcinoma, demonstrating good activity and acceptable safety
profile (Von Hoff et al., 2009). Moreover, a massive tumor regres-
sion has been reported in an adult patient with a plurimetastatic
PTCH-mutant medulloblastoma, even though disease restaging
revealed tumor progression associated with the onset of a resist-
ance-conferring SMO mutation (Rudin et al., 2009). Despite this
encouraging activity in tumors carrying mutations in Hh pathway
effectors, a comparable efficacy did not emerge against tumors
with wild-type Hh components. In fact, both a phase II rand-
omized study comparing chemotherapy with or without GDC-
0449 in metastatic colorectal cancer patients (Berlin et al., 2010)
and a placebo-controlled trial with GDC-0449 as maintenance
therapy in advanced ovarian cancer patients (Kaye et al., 2010)
failed to reach the primary endpoint. It is worth considering that
Hh pathway, when unaffected by somatic mutations, is activated
in a paracrine manner and, therefore, its inhibition could lead to

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Maugeri-Saccà et al. Targeting cancer stem cells
DNA-damaging agents. Beside GSIs, anti-DLL4 agents are in phase
I clinical development. Even though safety data are still unavail-
able, antibody-mediated inhibition of Notch pathway has been con-
nected in preclinical models with the onset of vascular tumors and
liver histopathological alterations such as sinusoidal dilation and
centrilobular hepatocyte atrophy (Li et al., 2010; Yan et al., 2010).
Liver gene expression of DLL4-treated mice revealed a significant
up-regulation of endothelium-specific genes, indicating that DLL4
signaling is crucial for maintaining endothelial cells in a quiescent
state. Similar alterations were noted with a Notch 1-specific inhibi-
tory antibody and the GSI dibenzazepine, thus indicating that these
changes represent a class effect of DLL4 inhibition. In summary,
optimal clinical development of Notch antagonists must take into
account the complexity of the pathway. In particular, the opposite
effects of Notch receptors observed in different tumor types raise
the need for more selective modalities of Notch inhibition.
rationale and StrategieS for targeting the wnt pathway
The canonical Wnt/β-catenin signaling is by far the best charac-
terized among Wnt pathways. This signaling is mainly regulated
at the level of β-catenin, a protein maintained at low cytoplasmic
concentration by a destruction complex. Upon activation of the
Wnt pathway, the inhibition of the β-catenin destruction com-
plex allows β-catenin to translocate to the nucleus, leading to the
expression of target genes involved in several cellular processes
encompassing proliferation, motility and stem cell maintenance
(Moon et al., 2002).
The role of the Wnt pathway in cancer has been recognized
since the Vogelstein’s “adenoma to carcinoma sequence,” which
identified mutations in adenomatosis polyposis coli (APC) gene
as a critical event during colorectal carcinogenesis. The most direct
evidence linking the Wnt pathway with CSCs comes from a recent
study demonstrating that high Wnt activity identifies the colon
CSC population (Vermeulen et al., 2010). Moreover, myofibroblast-
secreted factors instructed differentiated colon cancer cells to acti-
vate the β-catenin-dependent transcription, leading to the gain of
a stem-like phenotype. Although in a less direct way, the increased
nuclear level of β-catenin that characterizes the switch from chronic
phase to blast crisis in chronic myelogenous leukemia suggests the
influence of Wnt on leukemia stem cells (Jamieson et al., 2004).
A number of Wnt modulators or inhibitors have been identi-
fied (de Sousa et al., 2011). COX-2 inhibitors such as celecoxib
and rofecoxib seem to exert modulatory activity on Wnt through a
reduced production of prostaglandin E2, a molecule able to prevent
the degradation of β-catenin. c-MET inhibitors have been also
indicated as indirect Wnt antagonists. The rationale underlying the
anti-Wnt activity of these compound comes from the mitigation
of the activity of c-MET downstream machinery, which relieves
the inhibition on glycogen synthase kinase 3β, a key enzyme of
the destruction complex. Finally, high-throughput drug screen-
ing has allowed the identification of a variety of Wnt inhibitors
acting at various level of the pathway, including compounds sta-
bilizing key components of the destruction complex, transcrip-
tion factor antagonists and molecules inhibiting Wnt secretion.
However, the majority of direct Wnt antagonists are in preclinical
development, and only few of these have recently entered phase I
dose-finding studies.
properties. Notch 2 expression correlates with better prognosis in
breast cancer (Parr et al., 2004), while in mesothelioma counteracts
the pro-survival effects of Notch 1 (Graziani et al., 2008).
Breast cancer represents a benchmark for studying the influ-
ence of Notch pathway on CSCs. Consistent with this, a signifi-
cant reduction of mammosphere-forming efficiency has been
achieved with a gamma-secretase inhibitor (GSI) or a Notch
4-neutralizing antibody in ductal carcinoma in situ of the breast
(Farnie et al., 2007). Since a similar effect was seen with the EGFR
tyrosine kinase inhibitor gefitinib, it is conceivable that the dual
inhibition of Notch and EGFR may exert a synergistic effect. The
HER2 gene is amplified in approximately 20% of human breast
cancers and, more recently, has been implicated in breast CSCs
maintenance and expansion (Cicalese et al., 2009; Magnifico et al.,
2009). Evidence connecting Notch with HER2 comes from the
demonstration that the HER2 promoter contains Notch-binding
sequences, while Notch signaling activation has been documented
in HER2-overexpressing cells (Korkaya and Wicha, 2009). Notably,
Notch inhibition through small interfering RNA or a GSI impaired
mammosphere formation and determined HER2 down-regulation
(Magnifico et al., 2009). Finally, human breast cancer mammos-
pheres have been shown to acquire an hypoxia-resistant pheno-
type following Notch 3 induction by IL-6 (Sansone et al., 2007).
Since cancer cells cultured under hypoxic condition gain stem-like
properties (Li et al., 2009), it is conceivable that Notch pathway
and the hypoxia-sensing machinery dynamically cooperate in con-
trolling the CSC pool. Accordingly, GBM-SCs are characterized
by high Notch activity that confers resistance to ionizing radia-
tion, whereas Notch inhibition depleted GBM-SCs and reverted
radioresistance (Fan et al., 2010; Wang et al., 2010a). GBM-SCs
exposed to the differentiation-inducing agent retinoic acid under-
went both growth arrest and expression of lineage-specific dif-
ferentiation markers (Ying et al., 2011). Consistently, microarray
analysis of retinoic acid-treated cells revealed a molecular profile
characterized by the down-regulation of Notch components, while
the constitutive activation of Nic rescued GBM-SCs from retinoic
acid-induced differentiation and subsequent depletion. Finally,
the Notch pathway has been also implicated in colon cancer. In
this model, the inhibition of the DLL4 combined with irinotecan
has been associated with a reduction of KRAS-mutant colon CSCs
frequency (Fischer et al., 2011). Given that KRAS mutations are
detectable in approximately 40% of colorectal tumors and con-
fer resistance to anti-EGFR therapy, Notch antagonists should be
exploited for improving the management of the large segment of
patients unsuitable for cetuximab and panitumumab.
The inhibition of Notch pathway is undergoing early clinical
trials aimed at determining the safety profile and optimal schedule
of GSIs. Since hormone receptor-positive breast cancer relies on
Notch activation when hormonal signaling are pharmacologically
inhibited (Rizzo et al., 2008), different clinical trials are evaluating
the combination of GSIs with anti-estrogen therapy. Moreover, a
phase I study combining the GSI RO4929097 with carboplatin and
paclitaxel as neoadjuvant therapy in stage II and III triple-nega-
tive breast cancer patients is currently enrolling. Triple-negative
breast cancers harbor CSC-like characteristics and are deficient
in DNA repair (Perou, 2011). Thus, this study will provide valu-
able information given the potential synergism between GSIs and