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, 97106 (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).68 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 Kalles group.11 in brief, CsC clonal analysis, using the linear amplificationmediated PCr (lamPCr) labeling technique and serial xeno graft transplantation, can identify true stemcells with selfrenewal properties.12

Targeting embryonic signaling pathways

the first experimental evidence of CsCs came from lapidot et al.13 in the 1990s who identified leukemia stem cells capable of initiating human acute myeloid leukemia (aml) after transplantation into sCiD mice. the investi gators further revealed CsCs capable of selfrenewal and production of cancer progenitor cells using a limiting dilution analysis.14 this in vivo xenograft transplantation approach to identify cells with selfrenewal capability has subsequently been applied to identify CsCs in many solid

National Cancer Institute, Division of Cancer Treatment and Diagnosis, Cancer Therapy Evaluation Program, Investigational Drug Branch, EPN7131, 6130 Executive Boulevard, Rockville, Bethesda, MD 20852, USA (n. Takebe, P. J. Harris, S. P. ivy). PSI International, Inc., 6500 Rock Spring Drive, Suite 650, Bethesda, MD 20817, USA (R. Q. Warren). Correspondence to: S. P. Ivy ivyp@ctep.nci.nih.gov

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Key points
The stochastic model and cancer stem cell (CSC) model of tumorigenesis could be combined to help explain tumor relapse and metastasis DNA mutations, microenvironmental factors, and/or epithelial-to-mesenchymal transition may drive CSCs towards a metastatic phenotype Tumors composed of small populations of CSCs plus large numbers of bulk tumor cells may be particularly susceptible to combination drug regimens that target each cell population The potential for cross-talk among signaling pathways by CSCs opens new opportunities for designing combination drug regimens 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 Measurement of biologic effects of anti-CSC therapeutic regimens will remain a challenge until more-effective methods to identify CSCs in vivo or in vitro surrogate assays are improved
Negative regulatory region mAbs DLL4 mAbs Other ligand mAbs Active ligand Notch soluble receptor decoys

Neighboring signal-sending cell

Ligand degradation or recycling

Delta (DLL1, DLL3, DLL4) or Jagged (JAG1, JAG2) Extracellular space Notch ADAM/ TACE -Secretase inhibitors -Secretase modulators Nicastrin Presenilin APH-1 PEN-2 -Secretase complex

tumors. in 2004, Clarke and colleagues identified a subset of rare breast CsCs using a limiting dilution assay in non obese diabetic sCiD mice that were enriched in CD44+/ CD24/lin cells.15 thereafter, in vivo xenograft limiting dilution analyses were employed to demonstrate CsCs with tumorigenic activity in a variety of malignancies including brain,16 colon,1719 head and neck,20 pancreas,21,22 hepatocellular,23 melanoma,24 liver,25 lung,26 ovarian,27 prostate,28 bladder,29 and ewing sarcoma.30 owing to the very similar phenotypes of normal stem cells and CsCs, efforts to identify and isolate CsCs remain a challenge for drug development and treatment strat egies. in solid tumors, the identification and enrichment of CsCs is dependent on the presence of biomarkers (that is, CD133+, CD44+, CD24low, and CD166+). alternatively, some CsCs can be identified using flow cytometrybased functional assays such as aldehyde dehydrogenase1 (alDH1) expression and side population assays to enrich the CsC population from patient tumor explants.16,31,32 experimental models suggest that CsCs may originate from normal stemcells or normal progenitor cells.33,34 several leukemia mouse models have supported this hypothesis and examples from chronic myeloid leu kemia (Cml) clinical samples have indicated that Cml progenitor cells could acquire selfrenewal capabilities.35 nonetheless, tumors often develop and progress due to deregulated selfrenewal pathways.34 thus, targeting these deregulated embryonic signaling pathways holds promise in clinical therapeutic development.

Activated receptor Signal-receiving cell

Transmembrane portion of Notch S2 cleavage

NICD S3 cleavage

Small interfering RNA, micro RNA to target mRNAs Cytoplasm MAML1 inhibitor HAT MAML1 CSL Nucleus On/derepressed SKIP Notch target genes HES family Myc p21

Figure 1 | Notch signaling pathway inhibition. Activation of the Notch receptor occurs following binding of membrane-bound Delta or Jagged ligands during cell-tocell contact. Following absorption and proteolysis of the heterodimer Notch receptor (by ADAM and -secretase complex), a soluble fragmentthe NICDis released into the cytoplasm. The NICD translocates to the nucleus where it serves as a transcriptional activator of Notch-associated target genes, including HES, Myc and p21. Potential therapeutic inhibitors of Notch signaling target events such as -secretase complex proteolysis and transcriptional activation. Abbreviations: ADAM, A disintegrin and metalloproteinase; CSL, CBF1/Su(H)/Lag-1; DLL, deltalike ligand; HAT, histone acetyltransferase; MAML1, Mastermind-like 1; mAbs, monoclonal antibodies; NICD, Notch intracellular domain; SKIP, ski-interacting protein; TACE, TNF--converting enzyme.

notch signaling pathway notch signaling has a critical role in regulating cellto cell communication during embryogenesis, cellular proliferation, differentiation, and apoptosis. 36 notch signaling is also critical for normal hematopoiesis, breast development, colorectal epithelial maturation, immune regulation, and neural stem cell survival.37,38 mammalian membranebound notch ligands consist of two structur ally distinct families: Deltalike ligands (Dlls) 1, 3 and 4, and Jagged ligands 1 and 2 that interact with four trans membrane notch receptors (notch 14, Figure 1). the pairing of notch ligandreceptors results in coordinated communication between adjacent cells. the extracellular region of the notch receptor contains numerous epi dermal growth factorlike domains that mediate inter actions with notch ligands. affinity between the notch ligand and receptor depends on the extent of epidermal growth factor domain fucosylation by the Fringe proteins, that is, lunatic, radical, or manic.39 notch receptors exist as hetero dimers, consisting of noncovalently bound extracellular and transmembrane domains. once ligandreceptor binding occurs, the notch receptor undergoes a conformational change to expose a previously protected site to proteolytic cleavage by metalloprotease and secretase, releasing an extracellular and intracellular fragment, respectively.40 these catalytic steps cleave the intracellularmembrane domain and release the active notch intracellular domain (niCD) into the cytoplasm. niCD undergoes nuclear translocation and binding to the transcription initiation complex
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and core binding factor1 (CBF1), thus modulating notchspecific gene expression.
Investigational Notch pathway-targeting agents inhibition of secretasemediated notch cleavage is a primary focus for the development of targeted thera peutics. several pharmaceutical companies have devel oped secretase inhibitors (Gsis) that are in the early clinical development. two of these agents (ro4929097 [roche] and mK0752 [merck]) are in phase i testing after evaluating a variety of schedules to determine safe and tolerable administration regimens (table 1). the initial 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 secretase 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 helical 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 dominantnegative 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.
Table 1 | Experimental Notch inhibitors agents in development
-Secretase inhibitors: MK0752, RO4929097, PF03084,014, LY450139, BMS-708163 -Secretase modifiers:122 MPC-7869 MAML1 inhibitors:48,49 MAMLCSLNotch, antennapedia/dominantMAML Negative regulatory region monoclonal antibodies123 DLL4 monoclonal antibodies:45,124,125 OMP21M18, DLL4 antibody, DLL4 Notch soluble receptor decoys126,127
120,121

Target
Notch homologs Notch ligands Other -secretase substrates Substrates of -secretase

Mechanism of action
Inhibition of Notch cleavage by -secretase Inhibition of Notch cleavage by -secretase Interference with Notch nuclear co-activator MAML1 Interference with ligand-induced Notch subunit separation Interference with ligandreceptor interaction Interference with ligandreceptor interaction

Notch homologs Other nuclear transcription factors that target MAML1 Individual Notch receptors and other Notch ligands Specific for DLL4

Relatively specific for Notch homologs Potential pan-Notch inhibition

Abbreviations: DLL, Delta-like ligand; MAML, Mastermind-like.

Hedgehog signaling pathway the Hh signaling pathway controls tissue polarity, pat terning maintenance, and stemcell maintenance during
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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 HhPtch1 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 cells57the downstream target in the sonic Hh signaling pathwayis activated.58 Hh signaling may also have a
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Endoplasmic reticulum and golgi apparatus Hh Skn Hh-secreting cell Dispatched

HhN

Ligands (IHh, DHh, SHh)

HhN Smo antagonist Robotnikinin Activated Smo HPI-4

HhN COS Ptch CDO and brother of CDO -Arrestin Kif3A Gli1/2 HPI 1 Cyclin D, Cyclin E, Myc Gli1, Ptch, HIP Gli1/2 Nucleus Active Active SuFu Gli1/2/3 HPI-2/3

were sometimes needed to inhibit cell proliferation, indicating the potential for nonspecific effects. robotnikinin, a small molecule that binds the extra cellular sHh protein, has been isolated from small molecule microarraybased screens.64 targeting sHh ligands may be an interesting approach for prevent ing tumor relapse and metastasis. tumorderived sHh ligands directly activate signaling in stromal cells and the specificity of sHh targeting may circumvent the indian Hh inhibition caused by smo inhibitors, which potentially causes skeletal bone deformities in young children. identification of small synthetic molecules Hh Protein inhibitors (HPis) 14 are of interest. HPi1 inhibits Gli1/2 activation, HPi2 and HPi3 inhibits Gli2 activation, and HPi4 inhibits formation of cilia, when smo is activated, resulting in Gli transcription factor activation.65 emerging preclinical models have demon strated that Hh signaling can modulate the architecture of the stromal microenvironment and the smo inhibitor iPi926 (infinity Pharmaceuticals) can lead to improved access of chemotherapeutic agents.66
Clinical development of Hh pathway administration of GDC0449 (Genentech), a small molecule smo inhibitor, resulted in no doselimiting toxicities (Dlts) in patients with advanced solid tumors. Common adverse events included dysgeusia, hair loss, nausea, vomiting, anorexia, dyspepsia, weight loss, hyponatremia, and fatigue.67 the GDC0449 study in 33 patients with advanced BCC reported two complete responses and 16 partial responses.68 in addition, there are currently three companysponsored phase ii trials evaluating the efficacy of GDC0449 in patients with ovarian cancer in remission, advanced colorectal cancer, and advanced BCC. Four phase i and 10 phase ii clinical trials of GDC0449 are also being sponsored by the national Cancer institute and the Cancer therapy evaluation Program. these trials include pediatric and adult patients with recurrent medulloblastoma, advanced pancreatic tumor (in combi nation with gemcitabine), glioblastoma multiforme, gastric carcinoma (in combination with FolFoX [folinic acid, fluorouracil and oxaliplatin]), prostate carcinoma, soft tissue sarcoma, breast cancer, chondrosarcoma, multiple myeloma (in posttransplant patients), and smallcell lung cancer (in combination with cisplatin and etoposide). a trial investigating a novel combina tion therapy to inhibit both Hh and notch signaling pathways in advanced breast cancer and softtissue sar comas is underway. Bristolmyers squibb, in collabora tion with exelixis, has initiated two phase i clinical trials of a smallmolecule smo inhibitor (Bms833923, Xl139) in patients with advanced or metastatic solid tumors. this agent has been also tested in multiple myeloma in combination with lenalidomide, dexamethasone, and bortezommib, in smallcell lung cancer in combination with carboplatin and etoposide, and in metastatic gastric, gastroesophageal, and esophageal adenocarcinoma in combination with cisplatin and capecitabine. Clinical data from these trials are not yet available.
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Cytoplasm

Figure 2 | Hedgehog signaling pathway inhibition. In the inactive state, the absence of Hh leads to inhibition of Smo by the transmembrane receptor Ptch while Gli1/2 are phosphorylated and removed from the cytoplasm through proteosomal degradation. In the active state, Hh is secreted by an adjacent cell and binds to Ptch, allowing Smo activation. Gli1/2 are released from the Smo protein complex and translocate to the nucleus, leading to transcriptional activation of Hhassociated genes. New therapeutic agents have been developed that target Hh and Smo activation and downstream proteins, such as Gli. Abbreviations: COS, costal; Hh, Hedgehog; HIP, Hedgehog interacting protein; HPI, Hedgehog protein inhibitor; Ptch, Patched; Smo, Smoothened; SuFu, suppressor of fused.

key role in maintenance of CsCs in Cml.59 inhibition of Hh signaling by the inhibitor cyclopamine was shown to inhibit epithelialtomesenchymal transistion (emt) and metastases in pancreatic cancer cell lines.60
Preclinical agents targeting the Hh pathway Cyclopamine (11deoxojervine), the prototype of Hh pathwayspecific inhibitors, is a plantderived steroidal alkaloid that binds to and deactivates smo.61 a number of preclinical studies have been performed using this agent; however, cells without the smo receptor underwent apoptosis, therefore caution is needed when interpreting the results of possible offtarget effects. other synthetic small molecules that are more potent inhibitors of smo have been described.62,63 Furthermore, several natural and synthetic smallmolecule inhibitors of smo are being investigated preclinically for antitumor activity (table 2). However, these in vitro studies should be interpreted with caution because high concentrations of Hh antagonist
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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.
Table 2 | Experimental Hedgehog inhibitors agents in development
Cyclopamine61 (natural compound) Synthetic small molecules IPI92666 GDC-0449128 (Cur-61414) BMS833923 Robotnikinin64 PF04449913 LDE225 HPI 1465
129

Target
Smo

Mechanism of action
Antagonist

Smo Smo Smo Extracellular sHh Smo Smo Gli1/2 (HPI-1) Gli2 (HPI-2 and HPI-3) Cilia (HPI-4)

Antagonist Antagonist Antagonist Inhibitor of sHh Antagonist Antagonist Antagonist

Abbreviations: Hh, Hedgehog; HPI, Hedgehog protein inhibitor; sHh, Sonic hedgehog; Smo, Smoothened.

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 catenin (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 catenin 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 catenin from the cytoplasm to the nucleus results in interaction with members of the tcell factorlymphocyte 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
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cancer, and wilms tumor.8,58,7780 in addition, deregulated wnt signaling has been associated with CsC activity; specifically, cutaneous CsCs require catenin 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/catenin signaling by interrupting catenin 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 catenin and CBP, no interference with catenininduced 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 catenin and CBP intereaction to catenin 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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Wnt mAbs WIF-1 DKKs LRP inhibitors LRP Dvl CK1 P Dvl-P Axin stabilizing agents APC -Catenin degradation PP2A GSK3 -Catenin Axin Multiprotein destruction complex PAR-1 Dvl NSC 668036 Naked Wnt Fz sFRP Fz receptor antibody Small molecule inhibitors

ecadherin, a membranebound glycoprotein involved in the adherence of adjacent cells. the loss of ecadherin in primary tumor tissue has been linked with tumor meta stasis and poor prognosis.93,94 wnt, notch and Hh are all known inducers of emt along with niche factors, such as members of the transforming growth factor (tGF) family of cytokines.9598 interactions between tGF and the embryonic stemcell signaling pathways can also have an important role in maintaining the stemcelllike characteristics of emtinduced tumor cells.99 thus, tar geting embryonic signaling pathways may also inhibit emt and metastasis.

Chemoresistance of cancer stem cells

-Catenin

Cytoplasm -Catenin Nucleus ICG-001 p300 -Catenin Tcf/Lef BCL9 Pygo -Catenin Tcf/Lef Target genes Myc Cyclin D1 TCF-1 Migration NSAIDs PPAR- MMP-7 Adhesion PNU-74654 Axin-2 CD44 Cox2, etc.

CBP

Promotes cell differentiation

Promotes cell proliferation

Figure 3 | Wnt/-catenin signaling pathway inhibition. In the absence of Wnt signaling, cytoplasmic levels of -catenin are tightly regulated by a multiprotein destruction complex. -Catenin levels are kept low through phosphorylation, which leads to ubiquitinylation and subsequent proteosomal degradation. Binding of Wnt to the Fz receptors and LRP co-receptors allows -catenin to be released from the multiprotein destruction complex. The free -catenin is translocated to the nucleus where it acts together with either p300 or CBP as a transcriptional activator of Wnt-associated genes. Agents that inhibit Wnt/Fz binding and downstream events are in development. Abbreviations: CBP, cyclic AMP response element-binding protein; Fz, frizzled; LRP, low-density lipoprotein receptor-related protein; mAbs, monoclonal antibodies.

the high frequency of tumor relapse following therapy suggests the presence of residual CsCs that are resistant to conventional therapy.1 an increase in chemoresistant CD44+/CD24/low cells has been demonstrated in Her2 positive breast cancer following treatment with neo adjuvant chemotherapy.100 in this study, posttreatment biopsies were enriched in cells with tumorigenic poten tial, as demonstrated by their increased potential for mammosphere formation in vitro and increased capacity to form tumors in a xenograft model. several mechanisms can be involved in CsC chemo resistance and radioresistance, including expression of the atPbinding cassette superfamily of active drug transporters.101 members of this transmembrane super family act as unidirectional cellular pumps and have been linked with multidrug resistance.102 resistance to anti cancer drugs occurs as a result of reduced levels of drug accumulation within the CsCs. also, resistance to radia tion therapy can be caused by increased expression of freeradical scavengers by CsCs. these molecules reduce intracellular levels of reactive oxygen species following radiation therapy.103 shortlived reactive oxygen species can cause damage both to Dna and proteins, leading to cell death. By reducing levels of reactive oxygen species, resistant cells can evade the accumulation of cytotoxic effects of radiation therapy.103

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

Cross-talk among signaling pathways

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
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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.104106 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 factorB signaling.108 Crosstalk between
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notch and the Pi3K/akt pathways was reported in a variety of cell types.38,109 interactions between eGFr and notch signaling have been reported in breast cancer cell lines.110 treatment of these cells in vitro with gefitinib, an eGFr inhibi tor, resulted in decreased expression of both eGFr and notch1. notch1 knockdown with small interfering rna in glioma cell lines led to decreased eGFr mes senger rna and protein expression. 111 silencing of notch expression was also associated with decreased expression of p53, a known transcriptional activator of eGFr.112 eGFr is overexpressed in a large propor tion of gliomas;113 thus, combination therapies targeting both eGFr and notch may have clinical benefits for this patient population. 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 tGF and each of the embryonic signaling pathways. For example, tGFinduced emt could be blocked by rna silencing of either the Hey1 or Jag1 genes, suggest ing that there is crosstalk between tGF and notch pathways.116 tGFinduced 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.
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Table 3 | Experimental Wnt inhibitors agents in development

Wnt monoclonal antibodies Soluble Wnt receptor (Fz 8 cysteine-rich domain fused to the human Fc domain) antibody (decoy receptor) Fz receptor antibody NSC668036 FJ9 (disrupts Fz 7 and Disheveled) Thiazolidinedione PNU-74654 ICG-001 (cyclic AMP response element-binding protein -catenin) NSAIDs (e.g., Cox2 inhibitor) Av65 Artificial F-box protein Sulindac Inhibitors of Wnt response

Target
Wnt ligands

Mechanism of action
Ligand binding89,90

Fz receptors Disheveled PDZ domain -Catenin reverse nuclear transport -Catenin T-cell factor

Inhibition of ligand-receptor interaction130 Inhibition of disheveled PDZ domain, causing stabilization of -catenin degradation complex87,88 Transportation of -catenin from the nucleus131 Inhibition of protein-protein interactions resulting in decreased -catenin-dependent gene expression85,132,133 Mechanism unknown, possibly -catenin proteosomal degradation84,134137 Interacts with Axin2 to stabilize Axin proteins causing -catenin loss84 Inhibition of Wnt secretion84

Protein degradation process Axin2

Inhibitors of Wnt production

Abbreviation: Fz, Frizzled.

Porcupine

Conclusions

the combined stochasticCsC 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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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
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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
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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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