REvIEWS

Targeting cancer stem cells by inhibiting Wnt,

notch, and Hedgehog pathways
Naoko Takebe, Pamela J. Harris, Ronald Q. Warren and S. Percy Ivy
abstract | Tumor relapse and metastasis remain major obstacles for improving overall cancer survival,
which may be due at least in part to the existence of cancer stem cells (CSCs). CSCs are characterized by
tumorigenic properties and the ability to self-renew, form differentiated progeny, and develop resistance to
therapy. CSCs use many of the same signaling pathways that are found in normal stem cells, such as Wnt,
Notch, and Hedgehog (Hh). The origin of CSCs is not fully understood, but data suggest that they originate
from normal stem or progenitor cells, or possibly other cancer cells. Therapeutic targeting of both CSCs and
bulk tumor populations may provide a strategy to suppress tumor regrowth. Development of agents that
target critical steps in the Wnt, Notch, and Hh pathways will be complicated by signaling cross-talk. The role
that embryonic signaling pathways play in the function of CSCs, the development of new anti-CSC therapeutic
agents, and the complexity of potential CSC signaling cross-talk are described in this Review.
Takebe, N. et al. Nat. Rev. Clin. Oncol. 8, 97–106 (2011); published online 14 December 2010; doi:10.1038/nrclinonc.2010.196

Introduction
tumors are composed of a heterogeneous group of cells,
demonstrated by the fact that some tumor cell fractions
can support new growth in xenograft models, whereas
other cell fractions do not.1 traditionally, two models have
been proposed to explain tumor cell heterogeneity: the
stochastic model and the hierarchy model. the stochas­
tic model states that tumors arise as a biologically homo­
geneous group of cells, with functional heterogeneity
arising through random (that is, stochastic) events.2 in this
model, tumor initiation may occur in any cell as a result
of an accumulation of Dna mutations, epigenetic regula­
tion, and a permissive microenvironment.3 the stochastic
model suggests that all tumor cells have the potential to
become cancer stem cells (CsCs), given the appropriate
conditions. alternatively, the CsC (or hierarchy) model
suggests that tumors are composed of a heterogeneous
group of cells that have arisen from stem­like precursors.
as these tumor cells differentiate, they form a mixture
of cells with different biological and phenotypic charac­
teristics, forming a cellular hierarchy. at the apex of this
hierarchy are CsCs, which serve as the source of newly
formed tumor cells.
a third model, which blends characteristics of the
stochastic and CsC models, may provide a mechanism
for the formation of both primary and metastatic tumors.4
Chromosomal instability within the CsC population,
together with extrinsic environmental factors, may lead
to the appearance of CsC heterogeneity. self­renewing
CsCs, comprising a small minority of tumor cells, initi­
ated tumor growth and formed new progenitor and bulk
tumor cells in severe combined immunodeficient (sCiD)
competing interests
The authors declare no competing interests.
mice.5 CsCs use a variety of signaling pathways to undergo
self renewal and differentiation, including wnt, notch,
and Hedgehog (Hh).6–8 the slow growth rate and chemo­
resistant characteristics suggest that CsCs may survive
routine chemotherapy, only to reinitiate tumor growth at
a later point in time.5
Given the appropriate combination of Dna mutations
and environmental factors, a subpopulation of CsCs
might acquire metastatic properties. migratory CsCs may
become lodged in distant anatomical sites and continue
through self­renewal and asymmetric cell division, to
produce progenitor cells and bulk tumor cells.9 Certain
tumors, such as melanoma, exhibit high levels of CsCs
suggesting that the hierarchical model may not apply
for all tumors.10 in these cases, additional studies will
be required to determine frequencies of CsCs, perhaps
using assays such as serial dilution experiments in
combination with gene insertion clonality assays, similar
to that proposed by von Kalle’s group.11 in brief, CsC
clonal analysis, using the linear amplification­mediated National Cancer
Institute, Division of
PCr (lam­PCr) labeling technique and serial xeno­ Cancer Treatment and
graft transplantation, can identify true stem­cells with Diagnosis, Cancer
Therapy Evaluation
self­renewal properties.12 Program, Investigational
Drug Branch, EPN7131,
Targeting embryonic signaling pathways 6130 Executive
Boulevard, Rockville,
the first experimental evidence of CsCs came from Bethesda, MD 20852,
lapidot et al.13 in the 1990s who identified leukemia stem­ USA (n. Takebe,
P. J. Harris, S. P. ivy).
cells capable of initiating human acute myeloid leukemia PSI International, Inc.,
(aml) after transplantation into sCiD mice. the investi­ 6500 Rock Spring
gators further revealed CsCs capable of self­renewal and Drive, Suite 650,
Bethesda, MD 20817,
production of cancer progenitor cells using a limiting USA (R. Q. Warren).
dilution analysis.14 this in vivo xenograft transplantation
Correspondence to:
approach to identify cells with self­renewal capability has S. P. Ivy
subsequently been applied to identify CsCs in many solid ivyp@ctep.nci.nih.gov

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RevieWS

Key points tumors. in 2004, Clarke and colleagues identified a subset

of rare breast CsCs using a limiting dilution assay in non­
■ The stochastic model and cancer stem cell (CSC) model of tumorigenesis could
obese diabetic sCiD mice that were enriched in CD44+/
be combined to help explain tumor relapse and metastasis
CD24–/lin– cells.15 thereafter, in vivo xenograft limiting
■ DNA mutations, microenvironmental factors, and/or epithelial-to-mesenchymal
dilution analyses were employed to demonstrate CsCs
transition may drive CSCs towards a metastatic phenotype
with tumorigenic activity in a variety of malignancies
■ Tumors composed of small populations of CSCs plus large numbers of bulk
including brain,16 colon,17–19 head and neck,20 pancreas,21,22
tumor cells may be particularly susceptible to combination drug regimens that
target each cell population
hepatocellular,23 melanoma,24 liver,25 lung,26 ovarian,27
prostate,28 bladder,29 and ewing sarcoma.30
■ The potential for cross-talk among signaling pathways by CSCs opens new
opportunities for designing combination drug regimens
owing to the very similar phenotypes of normal stem­
cells and CsCs, efforts to identify and isolate CsCs remain
■ New experimental agents are being developed to block Wnt, Notch, and Hedgehog
signaling by CSCs, some of which are being tested in early clinical trials
a challenge for drug development and treatment strat­
egies. in solid tumors, the identification and enrichment
■ Measurement of biologic effects of anti-CSC therapeutic regimens will remain
of CsCs is dependent on the presence of biomarkers (that
a challenge until more-effective methods to identify CSCs in vivo or in vitro
surrogate assays are improved is, CD133+, CD44+, CD24low, and CD166+). alternatively,
some CsCs can be identified using flow cytometry­based
functional assays such as aldehyde dehydrogenase­1
■ Negative regulatory region mAbs (alDH1) expression and side population assays to enrich
Neighboring
signal-sending cell ■ DLL4 mAbs Ligand degradation
■ Other ligand mAbs or recycling
the CsC population from patient tumor explants.16,31,32
Notch soluble
experimental models suggest that CsCs may originate
Active receptor decoys from normal stem­cells or normal progenitor cells.33,34
ligand several leukemia mouse models have supported this
hypothesis and examples from chronic myeloid leu­
kemia (Cml) clinical samples have indicated that Cml
Delta (DLL1, DLL3, DLL4) progenitor cells could acquire self­renewal capabilities.35
or Jagged (JAG1, JAG2)
nonetheless, tumors often develop and progress due
■ γ-Secretase inhibitors
Extracellular space ■ γ-Secretase modulators to deregulated self­renewal pathways.34 thus, targeting
ADAM/ Nicastrin
these deregulated embryonic signaling pathways holds
Notch TACE Presenilin promise in clinical therapeutic development.
APH-1
PEN-2
notch signaling pathway
Activated Transmembrane γ-Secretase notch signaling has a critical role in regulating cell­to­
portion of Notch complex
receptor cell communication during embryogenesis, cellular
proliferation, differentiation, and apoptosis. 36 notch
Signal-receiving cell S2 cleavage signaling is also critical for normal hematopoiesis, breast
NICD
development, colorectal epithelial maturation, immune
S3 cleavage regulation, and neural stem cell survival.37,38 mammalian­
membrane­bound notch ligands consist of two structur­
Small interfering RNA,
micro RNA to target mRNAs ally distinct families: Delta­like ligands (Dlls) 1, 3 and 4,
and Jagged ligands 1 and 2 that interact with four trans­
Cytoplasm
MAML1 membrane notch receptors (notch 1–4, Figure 1). the
inhibitor pairing of notch ligand–receptors results in coordinated
Notch target genes communication between adjacent cells. the extracellular
HAT ■ HES family region of the notch receptor contains numerous epi­
MAML1 ■ Myc
■ p21 dermal growth factor­like domains that mediate inter­
CSL SKIP
actions with notch ligands. affinity between the notch
Nucleus ligand and receptor depends on the extent of epidermal
growth factor domain fucosylation by the Fringe proteins,
On/derepressed
that is, lunatic, radical, or manic.39 notch receptors exist
Figure 1 | Notch signaling pathway inhibition. Activation of the Notch receptor as hetero dimers, consisting of noncovalently bound
occurs following binding of membrane-bound Delta or Jagged ligands during cell-to- extracellular and transmembrane domains.
cell contact. Following absorption and proteolysis of the heterodimer Notch once ligand–receptor binding occurs, the notch
receptor (by ADAM and γ-secretase complex), a soluble fragment—the NICD—is receptor undergoes a conformational change to expose
released into the cytoplasm. The NICD translocates to the nucleus where it serves a previously protected site to proteolytic cleavage by
as a transcriptional activator of Notch-associated target genes, including HES, Myc metalloprotease and γ­secretase, releasing an extracellular
and p21. Potential therapeutic inhibitors of Notch signaling target events such as
and intracellular fragment, respectively.40 these catalytic
γ-secretase complex proteolysis and transcriptional activation. Abbreviations:
ADAM, A disintegrin and metalloproteinase; CSL, CBF1/Su(H)/Lag-1; DLL, delta-
steps cleave the intracellular­membrane domain and
like ligand; HAT, histone acetyltransferase; MAML1, Mastermind-like 1; mAbs, release the active notch intracellular domain (niCD) into
monoclonal antibodies; NICD, Notch intracellular domain; SKIP, ski-interacting the cytoplasm. niCD undergoes nuclear translocation
protein; TACE, TNF-α-converting enzyme. and binding to the transcription initiation complex

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and core binding factor­1 (CBF­1), thus modulating Table 1 | Experimental Notch inhibitors
notch­specific gene expression.
agents in development Target Mechanism of action

Investigational Notch pathway-targeting agents γ-Secretase inhibitors: 120,121 Notch homologs Inhibition of Notch
MK0752, RO4929097, Notch ligands cleavage by
inhibition of γ­secretase­mediated notch cleavage is PF–03084,014, LY450139, Other γ-secretase substrates γ-secretase
a primary focus for the development of targeted thera­ BMS-708163
peutics. several pharmaceutical companies have devel­ γ-Secretase modifiers:122 Substrates of γ-secretase Inhibition of Notch
oped γ­secretase inhibitors (Gsis) that are in the early MPC-7869 cleavage by
clinical development. two of these agents (ro4929097 γ-secretase
[roche] and mK0752 [merck]) are in phase i testing after MAML1 inhibitors:48,49 Notch homologs Interference with
evaluating a variety of schedules to determine safe and MAML–CSL–Notch, Other nuclear transcription Notch nuclear
tolerable administration regimens (table 1). the initial antennapedia/dominant–MAML factors that target MAML1 co-activator MAML1

testing of mK0752 in t­cell acute lymphoblastic leukemia
was disappointing due to the Gsi­associated toxicity of gut
goblet cell hyperplasia and associated secretory diarrhea
that was dose limiting. in a mouse model of t­cell acute
lymphoblastic leukemia, the co­administration of gluco­
corticoids and Gsi resulted in the successful reduction of
toxic effects in the gut, without impairing efficacy.41
strong evidence supports Gsi treatment of estrogen
receptor­positive breast cancer in which estrogen block­
ade by anti­estrogens or aromatase inhibitors leads to
dependence on notch signaling. rizzo and colleagues
found that estrogen inhibited notch activity was mediated
in part through inhibition of γ­secretase activity.42 the
study suggested that inhibition of notch signaling may be
a useful strategy in breast cancer. single­agent Gsi therapy
may also have activity in triple­negative breast cancer, as
this tumor type harbors CsC­like characteristics.
Dll4 is a notch ligand involved in the process of angio­
genesis. DLL4 mutations in mice result in severe disrup­
tion of the vasculature,43 whereas heterozygous deletion
of DLL4 is in general lethal.44 anti­Dll4 agents are in
phase i clinical development. no recommended phase ii
doses or schedules have been published. reported com­
plications of continuous antibody­mediated inhibition
of notch include the development of angiomas and neo­
vascular tuft formation.45,46 these effects may, however,
be antibody­specific.
agents that target mastermind­like (maml)–Csl–
notch complex formation, which is part of the notch
transcriptional complex, remain in very early preclinical
development. However, this approach to inhibit notch
transcription is unique as stapled α­helical peptides have
been used to target this complex.47,48 another interesting
approach is to use a genetically engineered fusion protein
of the Drosophila transcription factor antennapedia with
the truncated version of maml (antP/Dn maml)
that behaves in a dominant–negative fashion and inhib­
its notch activation.49 the fusion protein is internalized
and transported to the nucleus.49 selective inhibition of
notch receptors reduce intestinal toxic effects in rodent
xenograft models and may hold promise in future clini­
cal development.50 this strategy is appealing as it may
circumvent some of the off­target adverse events caused
by notch inhibition, such as diarrhea.
Hedgehog signaling pathway
the Hh signaling pathway controls tissue polarity, pat­
terning maintenance, and stem­cell maintenance during
Negative regulatory region Individual Notch receptors Interference with
monoclonal antibodies123 and other Notch ligands ligand-induced Notch
subunit separation
DLL4 monoclonal Specific for DLL4 Interference with
antibodies:45,124,125 ligand–receptor
OMP–21M18, DLL4 antibody, interaction
DLL4
Notch soluble receptor Relatively specific for Notch Interference with
decoys126,127 homologs ligand–receptor
Potential pan-Notch inhibition interaction
Abbreviations: DLL, Delta-like ligand; MAML, Mastermind-like.
embryonic development. 51 Hyperactivation of this
pathway, by either mutation or deregulation, has recently
been recognized to cause tumorigenesis in a wide variety
of tissues. most notably, the discovery of mutations
within the human homolog of the Drosophila patched
gene (Ptch1) in a rare hereditary form of basal cell carci­
noma (BCC), was the first clue to the involvement of
this pathway in patients with Gorlin syndrome.52,53 Hh
pathway involvement has been observed in other types of
non­mutation driven, paracrine deregulation of signal­
ing.54 the most interesting, and still evolving concept,
involves the association of Hh signaling with CsCs.
Hh is released from the cell through a dedicated
transmembrane transporter Dispatched after acyla­
tion of Hh n­terminus by the enzyme rasp located
in the endoplasmic reticulum.55 Binding of Hh to the
transmembrane receptor Ptch1 initiates signaling via
the Hh pathway (Figure 2). Ptch1 inhibits the receptor
smoothened (smo) by preventing its localization to the
primary cilium, a nonmotile projection present on most
vertebrate cells. in the presence of Hh, the Hh–Ptch1
complex is internalized, allowing smo activation.
localization of smo to the primary cilium, instead of
the plasma membrane, initiates a signaling cascade in
mammals, leading to the activation of the Gli family of
zinc­finger transcription factors. in vertebrates, there
are three Gli proteins: Gli1 serves to activate Hh target
genes, Gli2 acts both as an activator and repressor, and
Gli3 acts as a repressor of target­gene transcription. Hh
signaling seems to be dependent on the relative balance
of Gli activator and repressor forms.56 in breast CsCs,
Bmi­1, a transcriptional repressor of polycomb group
of transcription factors, and known to be a key regu­
lator of the self­renewal of normal and leukemic stem
cells57—the downstream target in the sonic Hh signaling
pathway—is activated.58 Hh signaling may also have a

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Endoplasmic
were sometimes needed to inhibit cell proliferation,
reticulum and golgi indicating the potential for nonspecific effects.
Hh
apparatus
Skn robotnikinin, a small molecule that binds the extra­
cellular sHh protein, has been isolated from small­
Hh-secreting cell
molecule microarray­based screens.64 targeting sHh
HhN ligands may be an interesting approach for prevent­
Dispatched ing tumor relapse and metastasis. tumor­derived sHh
ligands directly activate signaling in stromal cells and
the specificity of sHh targeting may circumvent the
HhN indian Hh inhibition caused by smo inhibitors, which
Ligands potentially causes skeletal bone deformities in young
(IHh, DHh, SHh) Smo antagonist children. identification of small synthetic molecules
Robotnikinin Hh Protein inhibitors (HPis) 1–4 are of interest. HPi­1
Activated HPI-4
Smo inhibits Gli1/2 activation, HPi­2 and HPi­3 inhibits Gli2
activation, and HPi­4 inhibits formation of cilia, when
HhN
smo is activated, resulting in Gli transcription factor
COS activation.65 emerging preclinical models have demon­
Ptch SuFu strated that Hh signaling can modulate the architecture
Gli1/2/3
of the stromal microenvironment and the smo inhibitor
CDO and brother of CDO β-Arrestin iPi­926 (infinity Pharmaceuticals) can lead to improved
HPI-2/3
Kif3A access of chemotherapeutic agents.66
Gli1/2 Active
HPI 1
Clinical development of Hh pathway
administration of GDC­0449 (Genentech), a small­
Cyclin D, Cyclin E, Myc molecule smo inhibitor, resulted in no dose­limiting
Cytoplasm
Gli1, Ptch, HIP toxicities (Dlts) in patients with advanced solid tumors.
Gli1/2 Active Common adverse events included dysgeusia, hair
loss, nausea, vomiting, anorexia, dyspepsia, weight loss,
Nucleus hyponatremia, and fatigue.67 the GDC­0449 study in
33 patients with advanced BCC reported two complete
Figure 2 | Hedgehog signaling pathway inhibition. In the inactive state, the absence responses and 16 partial responses.68 in addition, there
of Hh leads to inhibition of Smo by the transmembrane receptor Ptch while Gli1/2 are currently three company­sponsored phase ii trials
are phosphorylated and removed from the cytoplasm through proteosomal evaluating the efficacy of GDC­0449 in patients with
degradation. In the active state, Hh is secreted by an adjacent cell and binds to ovarian cancer in remission, advanced colorectal cancer,
Ptch, allowing Smo activation. Gli1/2 are released from the Smo protein complex and advanced BCC.
and translocate to the nucleus, leading to transcriptional activation of Hh-
Four phase i and 10 phase ii clinical trials of GDC­0449
associated genes. New therapeutic agents have been developed that target Hh and
Smo activation and downstream proteins, such as Gli. Abbreviations: COS, costal;
are also being sponsored by the national Cancer institute
Hh, Hedgehog; HIP, Hedgehog interacting protein; HPI, Hedgehog protein inhibitor; and the Cancer therapy evaluation Program. these
Ptch, Patched; Smo, Smoothened; SuFu, suppressor of fused. trials include pediatric and adult patients with recurrent
medulloblastoma, advanced pancreatic tumor (in combi­
nation with gemcitabine), glioblastoma multiforme,
key role in maintenance of CsCs in Cml.59 inhibition of gastric carcinoma (in combination with FolFoX [folinic
Hh signaling by the inhibitor cyclopamine was shown acid, fluorouracil and oxaliplatin]), prostate carcinoma,
to inhibit epithelial­to­mesenchymal transistion (emt) soft tissue sarcoma, breast cancer, chondrosarcoma,
and metastases in pancreatic cancer cell lines.60 multiple myeloma (in post­transplant patients), and
small­cell lung cancer (in combination with cisplatin
Preclinical agents targeting the Hh pathway and etoposide). a trial investigating a novel combina­
Cyclopamine (11­deoxojervine), the prototype of Hh tion therapy to inhibit both Hh and notch signaling
pathway­specific inhibitors, is a plant­derived steroidal pathways in advanced breast cancer and soft­tissue sar­
alkaloid that binds to and deactivates smo.61 a number comas is underway. Bristol–myers squibb, in collabora­
of preclinical studies have been performed using this tion with exelixis, has initiated two phase i clinical trials
agent; however, cells without the smo receptor underwent of a small­molecule smo inhibitor (Bms­833923, Xl139)
apoptosis, therefore caution is needed when interpreting in patients with advanced or metastatic solid tumors.
the results of possible ‘off­target’ effects. other synthetic this agent has been also tested in multiple myeloma in
small molecules that are more potent inhibitors of smo combination with lenalidomide, dexamethasone, and
have been described.62,63 Furthermore, several natural bortezommib, in small­cell lung cancer in combination
and synthetic small­molecule inhibitors of smo are being with carboplatin and etoposide, and in metastatic gastric,
investigated preclinically for antitumor activity (table 2). gastroesophageal, and esophageal adenocarcinoma in
However, these in vitro studies should be interpreted with combination with cisplatin and capecitabine. Clinical
caution because high concentrations of Hh antagonist data from these trials are not yet available.

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the safety of GDC­0449 was reported in a phase i clin­
ical trial of patients with metastatic or locally advanced
BCC. 68 of 33 evaluable patients, eight instances of
grade 3 adverse events were considered possibly attribu­
table to GDC­0449. these adverse events included
fatigue, hyponatremia, muscle spasm, and atrial fibrilla­
tion. no Dtls were observed and with the exception of
one grade 3 lymphopenia, andno hematologic adverse
events were observed.
other Hh pathway inhibitors are also in development.
For example, iPi­926 (infinity Pharmaceuticals), a cyclo­
pamine­derived inhibitor of the Hh pathway, is being
evaluated in clinical trials for advanced­stage solid tumors
and metastatic pancreatic cancer, and in a randomized,
double­blind, placebo­controlled phase ii study with gem­
citabine. PF­04449913 (Pfizer), a smo inhibitor, is cur­
rently being examined in a phase i study as a single agent
or in combination with dasatinib in patients with Cml. a
new smo antagonist lDe225 (novartis), is being investi­
gated in a phase i trial in patients with advanced solid
tumors, phase i study in BCC, and in a phase i pediatric
dose­finding study.
Wnt signaling pathway
wnt proteins consist of 19 highly conserved glycoproteins
that serve as ligands for the Frizzled (Fz) transmembrane
receptor.69 During embryogenesis, wnt proteins direct
cell fate determination at various stages of development
and their signaling acts to regulate the development of a
variety of organ systems including cardiovascular, central
nervous system, renal, and lung.70 in adults, wnt signal­
ing has a key role in the regulation of tissue self­renewal,
particularly in intestinal crypts, hair follicles, and bone
growth plates.71,72 loss­of­function and gain­of­function
studies in mice with β­catenin (CTNNB1) mutations
have demonstrated the importance of the wnt signaling
pathway in multiple organ systems.70
wnt binding to the Fz receptors initiates two distinct
signaling cascades, termed canonical or non­canonical.
the canonical pathway leads to the accumulation of
β­catenin in the nucleus and subsequent transcriptional
activation of targeted genes (Figure 3), but this does not
occur in the non­canonical pathway. to be functional,
wnts are lipid modified through palmitoylation. 73
secretion of wnt is carried out by the transmembrane
protein wntless.74 multiple wnt homologs have demon­
strated high­affinity binding to the conserved cysteine­
rich domain of Fz.75 interaction of the wnt proteins with
10 known mammalian Fz receptors76 suggests that the
potential for multiple wnt/Fz pairings may fine tune
the cellular response to wnt. translocation of β­catenin
from the cytoplasm to the nucleus results in interaction
with members of the t­cell factor–lymphocyte enhancer
factor family of transcriptional factors and subsequent
activation of wnt targeted genes.
Deregulated Wnt signaling in cancer or CSCs
aberrant wnt signaling has been reported in tumors from
patients with hepatocellular carcinoma, hepatoblastoma,
colorectal cancer, aml, Cml, multiple myeloma, gastric
nature reviews | clinical oncology
© 2011 Macmillan Publishers Limited. All rights reserved
RevieWS
Table 2 | Experimental Hedgehog inhibitors
agents in development Target Mechanism of action
Cyclopamine61 (natural compound) Smo Antagonist
Synthetic small molecules
IPI–92666 Smo Antagonist
GDC-0449128 (Cur-61414) Smo Antagonist
BMS–833923 129
Smo Antagonist
Robotnikinin64 Extracellular sHh Inhibitor of sHh
PF–04449913 Smo Antagonist
LDE225 Smo Antagonist
HPI 1–465 Gli1/2 (HPI-1) Antagonist
Gli2 (HPI-2 and HPI-3)
Cilia (HPI-4)
Abbreviations: Hh, Hedgehog; HPI, Hedgehog protein inhibitor; sHh, Sonic hedgehog; Smo, Smoothened.
cancer, and wilms tumor.8,58,77–80 in addition, deregulated
wnt signaling has been associated with CsC activity;
specifically, cutaneous CsCs require β­catenin signaling
to maintain their tumorigenic phenotype.81
a microarray analysis enriched from patients with
aml demonstrated that the wnt signaling pathway is
deregulated in leukemic stem cells when compared with
normal hematopoietic stem cells.82 in leukemic stem cells,
upregulation of genes encoding the wnt pathway pro­
teins axin and adenomatous polyposis coli are observed
frequently and deregulation of wnt signaling in aml
stem cells may contribute to leukemogenesis.83
Preclinical agents targeting the Wnt pathway
a number of experimental agents are currently being
evaluated for their ability to inhibit wnt signaling
(table 3). Chen et al.84 identified two new classes of
small molecules that inhibit wnt signaling. the first is a
membrane­bound acyltransferase that inhibits the acti­
vity of Porcupine, a molecule essential for wnt synthesis.
the second inhibits the destruction of axin, a suppres­
sor of wnt signaling activity. in addition, the small mol­
ecule iCG­001 (institute for Chemical Genomics), was
reported to selectively inhibit wnt/β­catenin signaling
by interrupting β­catenin binding to the transcriptional
cofactor cyclic amP response element­binding protein
(CBP).85 iCG­001 treatment of colon carcinoma cell lines
resulted in apoptosis, while sparing normal colonic epi­
thelial cells. although iCG­001 specifically blocked the
interaction between β­catenin and CBP, no interference
with β­catenin­induced p300 signaling was observed,
despite the high degree of sequence homology between
CBP and p300 (63%).85 iCG­001 seems to initiate the key
switch from β­catenin and CBP intereaction to β­catenin
and p300 interaction that controls a fundamental stem­
cell and progenitor cell switching, resulting in cell dif­
ferentiation. 86 several compounds are being tested
preclinically to target the PDZ domain of Disheveled,
nsC668036 and FJ9.87,88 Disheveled is a key protein in
the wnt signaling pathway that links extracellular signals
and downstream signals and may be able to inhibit both
non­canonical and canonical wnt signaling pathways.
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e­cadherin, a membrane­bound glycoprotein involved in

Wnt mAbs Small molecule
inhibitors the adherence of adjacent cells. the loss of e­cadherin in
primary tumor tissue has been linked with tumor meta­
WIF-1 sFRP
stasis and poor prognosis.93,94 wnt, notch and Hh are all
DKKs Wnt Fz receptor known inducers of emt along with niche factors, such as
antibody
LRP inhibitors Fz members of the transforming growth factor­β (tGF­β)
family of cytokines.95–98 interactions between tGF­β and
LRP
the embryonic stem­cell signaling pathways can also
Dvl NSC 668036
have an important role in maintaining the stem­cell­like
characteristics of emt­induced tumor cells.99 thus, tar­
CK1 P Naked
geting embryonic signaling pathways may also inhibit
Dvl-P Dvl emt and metastasis.
Axin stabilizing agents
PP2A
Axin Multiprotein
Chemoresistance of cancer stem cells
APC GSK3 PAR-1
destruction the high frequency of tumor relapse following therapy
β-Catenin
complex suggests the presence of residual CsCs that are resistant
β-Catenin
degradation to conventional therapy.1 an increase in chemoresistant
β-Catenin
CD44+/CD24–/low cells has been demonstrated in Her2­
positive breast cancer following treatment with neo­
Cytoplasm adjuvant chemotherapy.100 in this study, post­treatment
β-Catenin Target genes biopsies were enriched in cells with tumorigenic poten­
■ Myc tial, as demonstrated by their increased potential for
Nucleus ■ Cyclin D1
■ TCF-1 Migration
mammosphere formation in vitro and increased capacity
ICG-001
BCL9 Pygo
NSAIDs ■ PPAR-δ to form tumors in a xenograft model.
■ MMP-7 Adhesion
p300 β-Catenin PNU-74654 ■ Axin-2
several mechanisms can be involved in CsC chemo­
β-Catenin
CBP
■ CD44 resistance and radioresistance, including expression of
Tcf/Lef Tcf/Lef
■ Cox2, etc. the atP­binding cassette superfamily of active drug
transporters.101 members of this transmembrane super­
Promotes cell differentiation Promotes cell proliferation family act as unidirectional cellular pumps and have been
linked with multidrug resistance.102 resistance to anti­
Figure 3 | Wnt/β-catenin signaling pathway inhibition. In the absence of Wnt cancer drugs occurs as a result of reduced levels of drug
signaling, cytoplasmic levels of β-catenin are tightly regulated by a multiprotein accumulation within the CsCs. also, resistance to radia­
destruction complex. β-Catenin levels are kept low through phosphorylation, which tion therapy can be caused by increased expression of
leads to ubiquitinylation and subsequent proteosomal degradation. Binding of Wnt
free­radical scavengers by CsCs. these molecules reduce
to the Fz receptors and LRP co-receptors allows β-catenin to be released from the
intracellular levels of reactive oxygen species following
multiprotein destruction complex. The free β-catenin is translocated to the nucleus
where it acts together with either p300 or CBP as a transcriptional activator of radiation therapy.103 short­lived reactive oxygen species
Wnt-associated genes. Agents that inhibit Wnt/Fz binding and downstream events can cause damage both to Dna and proteins, leading to
are in development. Abbreviations: CBP, cyclic AMP response element-binding cell death. By reducing levels of reactive oxygen species,
protein; Fz, frizzled; LRP, low-density lipoprotein receptor-related protein; mAbs, resistant cells can evade the accumulation of cytotoxic
monoclonal antibodies. effects of radiation therapy.103

Cross-talk among signaling pathways

monoclonal antibodies against wnts, Fz receptor, or
secreted Fz­related protein have been tested to inhibit
wnt signaling at the extracellular level, with promising
antitumor activity.89,90
Epithelial-to-mesenchymal transition
emt originally defined a process of cellular reorganiza­
tion essential for embryonic development. this multi­step
process results in the loss of cell­to­cell adhesive prop­
erties, loss of cell polarity, and the gain of invasive and
migratory mesenchymal properties.91 During embryo­
genesis, emt allows progenitor cells to migrate to distant
sites within an embryo to form new tissue.92 the emt
process is reversible, with mesenchymal­to­epithelial
transition allowing cells with mesenchymal characteris­
tics to revert to epithelial­like cells. the emt processes
also occur during tumorigenesis, allowing some CsCs
to become metastatic. During emt, cells downregulate
instances of cross­talk between the embryonic signaling
pathways notch, wnt, or Hh and other signaling path­
ways have been reported in a variety of cell types.104–106
although aberrant activation of an individual pathway
may result in tissue specific carcinogenesis, these path­
ways rarely operate in isolation. Cross­talk between
signaling pathways has the potential to profoundly add
to the complexity of cellular responses to external stimuli.
a link between murine mammalian target of rapamycin
(mtor) and notch signaling pathways shows that cells
with constitutively activated mtor also express ele­
vated levels of niCD and Hes1, suggesting that notch
is upregulated by mtor.107 treatment with the mtor
inhibitor rapamycin led to decreased signaling of both
mtor and notch. upregulation of notch by mtor acts
through the stat3/p63/Jagged pathway. Conversely,
downregulation of notch signaling inhibited mtor, akt,
and nuclear factor­κB signaling.108 Cross­talk between

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 RevieWS

notch and the Pi3K/akt pathways was reported in a Table 3 | Experimental Wnt inhibitors
variety of cell types.38,109
agents in development Target Mechanism of action
interactions between eGFr and notch signaling have
been reported in breast cancer cell lines.110 treatment Wnt monoclonal antibodies Wnt ligands Ligand binding89,90
Soluble Wnt receptor (Fz 8
of these cells in vitro with gefitinib, an eGFr inhibi­ cysteine-rich domain fused to the
tor, resulted in decreased expression of both eGFr and human Fc domain) antibody
notch­1. notch­1 knockdown with small interfering (decoy receptor)
rna in glioma cell lines led to decreased eGFr mes­ Fz receptor antibody Fz receptors Inhibition of ligand-receptor
senger rna and protein expression. 111 silencing of interaction130
notch expression was also associated with decreased NSC668036 FJ9 (disrupts Fz 7 Disheveled PDZ Inhibition of disheveled PDZ
expression of p53, a known transcriptional activator and Disheveled) domain domain, causing stabilization of
β-catenin degradation complex87,88
of eGFr.112 eGFr is overexpressed in a large propor­
tion of gliomas;113 thus, combination therapies targeting Thiazolidinedione β-Catenin reverse Transportation of β-catenin from
both eGFr and notch may have clinical benefits for this nuclear transport the nucleus131

patient population. PNU-74654 β-Catenin Inhibition of protein-protein

an inverse correlation has been observed between
notch and Her2 signaling. Breast cancer cell lines that
overexpress Her2 had 6­fold to 20­fold lower levels of
notch transcriptional activity than cells expressing low
levels of Her2, suggesting that Her2 overexpression acts
to suppress notch signaling.114 By contrast, trastuzumab­
mediated suppression of Her2 phosphorylation resulted
in increased notch nuclear accumulation and increased
notch expression. alternative notch signaling may occur
in trastuzumab­resistant breast cancer cells. an analysis
of 48 primary breast tumors revealed a significant link
between notch 1 and 3 expression and Her2­negative
tumors.115 expression of notch in Her2­negative breast
tumors may represent a novel target for antitumor strat­
egies. Cross­talk between the notch and Her2 pathways
in breast cancer may provide cells with a mechanism for
trastuzumab resistance.
reports indicate that cross­talk occurs between
tGF­β and each of the embryonic signaling pathways.
For example, tGF­β­induced emt could be blocked by
rna silencing of either the Hey1 or Jag1 genes, suggest­
ing that there is cross­talk between tGF­β and notch
pathways.116 tGF­β­induced emt was observed in epi­
thelial cells. the cellular process of acquiring a metastatic
phenotype was mediated through the induction of the
notch ligand Jagged­1 and the notch target gene Hey1
in epithelial cells.117
Cross­talk between the wnt and Hh signaling pathways
can also occur through sFrP­1, expression of which is
induced by Gli1 in gastric cancer cell lines.118 Hh signaling
was found to inhibit wnt signaling through upregulation
of sFrP­1.118 interaction with Hh signaling and notch
may be important in breast cancer as Hh may modulate
the tumorigenic property in cooperation with notch.
Hh signaling activation induces Jagged 2, a notch ligand
resulting in upregulation of the notch pathway. this
interaction is also potentiated by active eGFr signaling as
described above. signaling pathways, instead of acting
as isolated units, may interact through cross­talk as part
of a broader signaling network. these cross­talk inter­
actions may contribute to the cellular diversity associated
with stem cells during embryogenesis and tissue main­
tenance. the identification of specific cross­talk net­
works in tumor cells may also allow more effective use of
combination antitumor therapies.
ICG-001 (cyclic AMP response T-cell factor interactions resulting in
element-binding protein β-catenin) decreased β-catenin-dependent
NSAIDs (e.g., Cox2 inhibitor) gene expression85,132,133
Av65 Protein Mechanism unknown, possibly
Artificial F-box protein degradation β-catenin proteosomal
Sulindac process degradation84,134–137
Inhibitors of Wnt response Axin2 Interacts with Axin2 to stabilize
Axin proteins causing β-catenin
loss84
Inhibitors of Wnt production Porcupine Inhibition of Wnt secretion84
Abbreviation: Fz, Frizzled.
Conclusions
the combined stochastic–CsC model provides a possible
explanation for the initiation of tumorigenesis, tumor
relapse, and metastasis in the majority of tumors. this
model suggests that CsCs evolve into cells with meta­
static potential through accumulated somatic mutations,
emt, or stromal microenvironmental factors. tumors
composed of small populations of CsCs intermixed with
large numbers of bulk tumor cells may require alterna­
tive treatment strategies to effectively inhibit growth and
prevent relapse. For this reason, CsCs are increasingly
becoming priority targets for the development of new
antitumor therapeutics.
these new approaches to cancer therapy will be accom­
panied by significant challenges. First among these is the
apparent lack of universal stem­cell­specific markers
to identify CsCs from different tissues. Putative CsCs
were successfully enriched using cell surface phenotype
in some preliminary pioneering work in this area, but
more recent data have demonstrated that CsCs undergo
dynamic changes at the level of cell­surface markers.119
these changes are possibly due to epigenetic regulation
and growth factors controlled by the microenvironment,
making it difficult to define the CsC subpopulation on
the basis of cell­surface marker expression. the identifi­
cation of CsC­associated markers and pharmaco­
dynamic marker assays to monitor the therapeutic effects
in patients are critical hurdles. ideally, in vivo imaging
of CsCs would be of interest to monitor the biological
effects of experimental agents, but these screens are not
yet available.
signaling pathways can interact through cross­talk as
part of broad, complex signaling networks. Cross­talk

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© 2011 Macmillan Publishers Limited. All rights reserved
RevieWS

may also contribute to the cellular diversity associated
with stem cells during embryogenesis and tissue main­
tenance, and may have a key role in the growth of CsCs.
the identification of cross­talk networks can provide new
opportunities for designing more­effective treatment
regimens. new experimental agents are being developed
to inhibit the wnt, notch, and Hh signaling pathways,
which are frequently utilized by stem cells. Proof­of­
concept clinical trials are currently underway for many
of these agents, including GDC­0449, ro4929097, and
several Hh and notch pathway inhibitors. although still
early in development, this new approach of targeting
both the bulk tumor and CsC populations may provide
a more­effective way of inhibiting tumor relapse and
metastasis. targeting of embryonic signaling pathways,
used by CsCs, represents an emerging and potentially
beneficial new field of cancer therapeutics.
Review criteria
Data for this Review were compiled by searching PubMed,
Google Scholar, and Clinicaltrials.gov databases. The
search terms included “Notch”, “Wnt”, “Hedgehog”,
“cancer stem cells”, “NOD-SCID”, “embryonic signaling
pathway”, “self-renewal”, “EMT”, “migrating cancer stem
cells”, “γ-secretase inhibitor”, “TGF-β”, and “BMP”. In
general, we selected full text articles published in English.

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author contributions
N. Takebe, P. J. Harris, R. Q. Warren and S. P. Ivy
contributed equally to the literature review,
discussions of the content, writing and reviewing and
editing of this manuscript before submission.
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