nature review cancer 2013

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WNTs are a family of 19 secreted glycoproteins1 thathave crucial roles in the regulation of diverse processes,including cell proliferation, survival, migration andpolarity, specification of cell fate, and self-renewal instem cells. Perturbation of the levels of WNT ligands,or altered activities of the proteins that are necessary forWNT signal transduction, can result in defects in embry-onic development; additionally, abnormal WNT signal-ling in adults may contribute to disease aetiology. A rolefor WNTs in cancer was first described three decadesago in mouse models of mammary cancer and in humanand mouse colon cancer. Aberrant overexpression ofWNT1 induced by a proviral insertion at the Wnt1 (alsoknown as int1) locus induces spontaneous mammaryhyperplasia and tumours in mice2,3, and Wnt1 transgenicmice similarly develop mammary tumours, suggesting acausative role for WNT1 in mammary tumorigenesis4.Further studies found that WNT1 and other WNTspromoted the stabilization of free pools of β-catenin(CTNNB1)5 and the activation of CTNNB1-dependent

transcription.Shortly after the characterization of WNT1 in mouse

models of mammary cancer, other studies pointed to acrucial role for hyperactivated WNT–CTNNB1 signal-ling in colorectal cancer6,7. Inherited inactivating muta-tions in adenomatous polyposis coli ( APC ) — which isa negative regulator of CTNNB1 stability (FIG. 1) — arefound in patients with familial adenomatous polyposis(FAP), which can progress to colorectal carcinomas fol-lowing concomitant activating mutations in KRAS andinactivating mutations in TP53 (reviewed in REFS 6,7).Both APC and CTNNB1 are also frequently mutatedin colorectal cancers of non-FAP patients 8,9, and

overexpression of constitutively active CTNNB1 or loss ofAPC function (both of which lead to hyperactivationof WNT–CTNNB1 signalling) can result in colorectaltumorigenesis in mouse models6,7. These observationsdemonstrate that mutations leading to the unregulatedactivation of WNT–CTNNB1 signalling contribute totumorigenesis in the colon.

It is now clear that WNTs modulate both CTNNB1-dependent (often referred to as ‘canonical’) WNTsignalling (FIG. 1) and CTNNB1-independent (oftenreferred to as ‘non-canonical’) WNT signalling path-ways (FIG. 2). The precise mechanisms by which a WNTstimulates CTNNB1-dependent versus CTNNB1–independent cellular responses are not fully elucidated,but probably involve the stimulation of distinct WNTreceptors. Reported transmembrane WNT receptorsinclude the ten members of the frizzled (FZD) familyof G-protein-coupled receptors (GPCRs), as well as thereceptor tyrosine kinases (RTKs) ROR1 and ROR2 andthe RTK-like protein RYK10,11. Although it is beyond the

scope of the present Review, it is worth noting that des-ignating WNT signalling pathways as either canonicalor non-canonical has some utility for enabling discus-sion, but the reality is that WNT ligands probably stim-ulate complex, non-linear networks that share manydownstream effectors.

In this Review we present evidence that the activity ofWNT signalling networks can unexpectedly correlate ineither a positive or negative manner with patient outcomesin different types of cancer. We further review evidencethat WNT signalling can either promote or inhibit tumourinitiation, growth, metastases and drug resistance in acancer-stage-specific and a cancer-type-specific manner.

1Institute for Stem Cell and

Regenerative Medicine,2Department of

Pharmacology and Howard

Hughes Medical Institute,3Molecular and Cellular

Biology Graduate Program

and 4University of

Washington School of

Medicine, University of

Washington, Seattle,

Washington 98109, USA.

Correspondence to R.T.M.

e-mail: [email protected]

doi:10.1038/nrc3419

Constitutively active

CTNNB1

Various forms of β-catenin

(CTNNB1) with either

amino-terminal truncations or

point mutations that prevent

phosphorylation and

degradation by theproteasome.

WNT signalling pathways astherapeutic targets in cancer

Jamie N. Anastas1,2,3,4 and Randall T. Moon1,2,4

Abstract | Since the initial discovery of the oncogenic activity of WNT1 in mouse mammary

glands, our appreciation for the complex roles for WNT signalling pathways in cancer has

increased dramatically. WNTs and their downstream effectors regulate various processes

that are important for cancer progression, including tumour initiation, tumour growth, cell

senescence, cell death, differentiation and metastasis. Although WNT signalling pathwayshave been difficult to target, improved drug-discovery platforms and new technologies have

facilitated the discovery of agents that can alter WNT signalling in preclinical models, thus

setting the stage for clinical trials in humans.

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NATURE REVIEWS | CANCER VOLUME 13 | JANUARY 2013 | 11

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mailto:[email protected]



Our first goal is to paint a more complex picture than thewidely held presumption that elevated WNT–CTNNB1signalling in cancer consistently leads to negative clini-

cal outcomes. Our second goal is to emphasize poten-tial roles for CTNNB1-independent WNT signalling incancer. It is our hope that appreciating the complexity ofWNT signalling in different contexts will facilitate thedevelopment of improved therapies.

Aberrations in WNT signalling in cancer

The high frequency of WNT pathway mutations in manydifferent cancers underscores the importance of WNT–CTNNB1 signalling to carcinogenesis. In addition to APC mutations, sequencing of patient colorectal tumoursby The Cancer Genome Atlas Network and others hasidentified mutations in other WNT pathway genes suchas transcription factor 7-like 2 (TCF7L2; previouslyknown as TCF4), CTNNB1 and Wilms tumour gene onthe X chromosome (WTX ; also known as FAM123B) thatare predicted to activate WNT–CTNNB1 signalling 12,13.Both missense mutations and other mutations that arepredicted to disrupt the phosphorylation and degra-dation of CTNNB1 are also frequent in hepatocellularcarcinoma (HCC)14,15, medulloblastoma16 and ovariancancer17, whereas deletions and truncation mutations in AXIN1 are common in HCC and colorectal tumours18,19 (TABLE 1). We note that most WNT pathway mutationsthat are observed in cancer result in hyperactivation ofWNT–CTNNB1 signalling. Although mutations in genessuch as FZD4, LRP5 and LRP6 that are thought to inhibit

WNT signalling have been identified in other disorders(including coronary artery disease and neurodegenera-tive disorders20–22), similar WNT-pathway inactivatingmutations have not been characterized in cancer.

WNTs and WNT pathway components are also fre-quently over- or under-expressed in different humancancers (TABLE 1), and these changes in expression profilesoften correlate with epigenetic activation or inactivationof gene promoters23–26. The expression patterns of WNTsignalling components can serve as a prognostic indica-tor of patient outcomes (TABLE 2, Supplementary infor-mation S1 (table)). As predicted by the seminal studieslinking WNT–CTNNB1 signalling to carcinogenesis of

the breast and colon, high levels of nuclear CTNNB1,which are normally interpreted as a sign of increasedWNT–CTNNB1 signalling activity, correlate withpoor prognosis in these cancers27–35. However, recentstudies suggest that high levels of WNTs and nuclearCTNNB1 do not always predict poor prognosis.In medulloblastoma, patients with activating muta-tions in CTNNB1 had greater disease-free survival thanpatients with mutations in the sonic hedgehog (SHH)pathway, which probably reflects the unique cell-typeorigins of medulloblastomas that are driven by these dif-ferent mutations36,37. Elevated levels of nuclear CTNNB1also correlate with improved patient outcomes in malig-nant melanoma, ovarian cancer and prostate cancer 38–40.Although information regarding CTNNB1-independentWNT signalling pathways in cancer is still limited, sev-eral studies suggest that increased expression of WNT5A(which leads to CTNNB1-independent signalling) cor-relates with poor clinical outcomes in melanoma41–43 andgastric cancer44, yet correlates with improved patientsurvival in breast45 and colon cancers46. Importantly,

some of these studies have not yet been independentlyconfirmed. Furthermore, it is not always clear which ofthese aberrations in WNT signalling are causativeof poor clinical outcomes in patients with cancer, andwhich are merely correlative. Despite these importantcaveats, the findings indicating that altered expressionof WNT signalling components can be predictive ofeither improved or worsened patient outcomes highlightthe need to better understand the context-dependentroles for WNT signalling in cancer, which is furtherdiscussed below.

WNT signalling and growth controlWNTs. Although different cancer cell types vary dra-matically in their growth responses to stimulation bydifferent WNT ligands, it is clear that autocrine WNTsignalling has a crucial role in the growth and survival of

various cancer cells. For example, early studies of mam-mary epithelial cells revealed that WNT7A, WNT3Aand WNT1 efficiently transform these cells, whereasWNT6, WNT4 and WNT5A do not47,48. Similarly, over-expression of WNT1, but not WNT7B or WNT5A,induced hyperplasia in mouse mammary cells grownin vivo in mammary fat pads49.

Other cancer subtypes also exhibit unique sensitivitiesand responses to different WNT ligands. WNT3A pro-motes the stabilization of CTNNB1 and the activation of

transcription that is dependent on the TCF/LEF familyof transcription factors in both myeloma and prostatecancer cell lines50–53. Furthermore, the expression ofconstitutively active CTNNB1 is sufficient to enhancethe growth of myeloma cells in vitro51 and of pros-tate tumours in mouse models54,55. This suggests thatWNT3A–CTNNB1 signalling promotes the growth ofthese cancers. Consistent with the finding that activa-tion of WNT–CTNNB1 signalling can be either posi-tively or negatively correlated with cancer progression,WNT3A–CTNNB1 signalling can conversely inhibitthe growth of some cancers. For example, elevated lev-els of WNT3A significantly decrease the growth of both

At a glance

• WNTs are secreted glycoproteins that regulate multiple signalling pathways through

both β‑catenin (CTNNB1)‑dependent and CTNNB1–independent mechanisms.

• The activation of WNT signalling pathways can be both positively and negatively

correlated with patient outcomes in different types of cancer.

• WNT–CTNNB1 signalling can either promote or inhibit tumour initiation,

growth, metastases and drug resistance in a cancer‑stage‑specific and a

cancer‑type‑specific manner.• CTNNB1‑independent WNT signalling pathways also contribute to tumorigenesis

and cancer progression in a context‑dependent manner.

• Aberrations in WNT signalling pathways and alterations in other oncogene and

tumour suppressor pathways cooperate to drive cancer initiation and progression.

• Multiple strategies for targeting WNT signalling — ranging from small molecules

to blocking antibodies, and peptide agonists and antagonists — are now in

development, thus paving the way for initial clinical trials using WNT modulators

in cancer patients.

R E V I E W S

12 | JANUARY 2013 | VOLUME 13 www.nature.com/reviews/cancer


http://www.nature.com/nrc/journal/v13/n1/suppinfo/nrc3419.html






a b

DKK

LRP5 or LRP6

FZD WAY-316606

WNT

WNT

WIFIWP

SFRP

DVL DVL

Lithium

CT99021

BIO

GSK3

Pyrvinium

IC261

CK1

APC

CTNNB1

AXIN

APC

CTNNB1

CTNNB1

CTNNB1

CTNNB1

CTNNB1CTNNB1

AXIN

BCL9p300

WTX

TRCP

TNKS

XAV939

JW55

SKL2000

ICG-001

3289-8625

PEN-N3

FJ9

NSC668036

NC043

BC21

PNU-7465431

PKF115-584

CGP049090

PKF118-310

2TG

STG28

TLE HDACTCF/LEF

TCF/LEF

P

P P

P

P

P

PP

LRP5 or LRP6

FZD

Ub

UbUb

Ub Ub

Ub

Ub Ub

Ub

GSK3

CK1

Nucleus Nucleus

human and mouse melanoma cells grafted in mice, whileactivating CTNNB1-dependent transcription40,56. Whymight WNTs such as WNT3A and WNT7A promotegrowth in some cancers but not in others? One possible

explanation is that WNTs activate different signallingpathways depending on the cellular context. WNT7Aregulates both proliferation and CTNNB1-dependenttranscription in ovarian cancer cells57, but in leukaemiccells58 WNT7A inhibits proliferation yet has little effecton CTNNB1-dependent transcription. It is possible thatin leukaemic cells WNT7A regulates different signallingpathways that are important for growth inhibition. Inendometrial carcinoma cells, cotransfection of WNT7Aand FZD5 promotes CTNNB1-dependent transcrip-tion, whereas cotransfection of WNT7A and FZD10activates JUN N-terminal kinases (JNKs)59. These datasuggest that WNT7A can regulate different signalling

cascades depending on the combination of receptors thatare expressed by a particular cancer type. Sometimes thedivergent responses of cancer cells to ligand stimulationdo not seem to involve the activation of unique signal-

ling pathways, but reflect intrinsic differences in cellularinterpretations of WNT–CTNNB1 signalling. For exam-ple, in melanoma cells WNT3A promotes the expressionof genes associated with melanocyte differentiation anddecreases the expression of genes associated with pro-liferation40, yet in prostate cancer cells WNT3A inducesthe expression of genes that are important for growth andsurvival55,60. WNT-dependent stabilization of CTNNB1alters the transcriptional profiles of many different targetgenes in a tissue-type-specific manner, and these expres-sion changes involve crosstalk with other transcriptionfactors and cofactors that are differentially expressed incancers (BOX 1).

Figure 1 | The WNT–CTNNB1 signalling pathway. β-catenin (CTNNB1)-dependent WNT signalling pathways have crucial

roles in the regulation of diverse cell behaviours, including cell fate, proliferation, survival, differentiation, migration and

polarity. Recently, numerous studies have identified small-molecule inhibitors and activators of various pathway

components; these are indicated in this figure in blue boxes. For a more detailed description of these inhibitors refer to

TABLE 3 and Supplementary information S2 (table).a | In the absence of WNT stimulation, a destruction

complex — containing the proteins adenomatous polyposis coli (APC), glycogen synthase kinase 3β (GSK3β) and

AXIN — phosphorylates (P) and targets CTNNB1 for ubiquitylation (Ub) and proteasomal degradation. In the absence of

WNTs, members of the TCF/LEF family of high-mobility-group transcription factors associate in a repressive complex with

transducin-like enhancer protein (TLE; also known as Groucho) co-repressor proteins, which promote the recruitment of

histone deacetylases (HDACs) to repress CTNNB1 target genes. b | The binding of WNTs, such as WNT3A and WNT1, tofrizzled (FZD) and LRP5 or LRP6 co-receptors transduces a signal across the plasma membrane that results in the activation

of the Dishevelled (DVL) protein. Activated DVL inhibits the destruction complex, resulting in the accumulation of CTNNB1,

which then enters the nucleus where it can act as a co-activator for TCF/LEF-mediated transcription. CTNNB1 acts

a transcriptional switch, as the presence of CTNNB1 reduces the association of TLE with TCF/LEF, while recruiting various

transcriptional cofactors including BCL9, Pygopus and histone acetyltransferases. WNT–CTNNB1-dependent transcription

ultimately modulates changes in cell behaviours such as proliferation, survival and differentiation. CK1, casein kinase 1;

DKK dickkopf homologue; SFRP, secreted frizzled-related protein; TNKS, tankyrase; βTRCP, β-transducin repeat-containing

E3 ubiquitin protein ligase; WIF, WNT inhibitory factor; WTX, Wilms tumour gene on the X chromosome.

R E V I E W S





WNT

SRC JNK

WNTWNT WNT

WIF WAY-316606

Foxy-5

NSC668036

FJ9

3289-8625

PEN-N3

SFRP

DVL

DVL

JNK

PKCCAMKII

RAC

PRICKLE

PLC

DAAM1

RHOA

CDC42

SCRIB

GIT

AP-1NFAT

PAK

ARHGEF7

FZDRYK ROR2

CELSR

PTK7 VANGL

Cytoskeleton

Ca2+

Ca2+

Ca2+

Nucleus

Feeder cells

Additional cells used in

co-culture experiments that

are intended to support the

growth of the cells of interest.

Increasing or decreasing CTNNB1-independentWNT signalling mediated by the altered expression ofWNT5A and WNT11 can also result in profound effectson cancer cell proliferation. WNT5A can act as a growthsuppressor in many cancers, including ovarian andthyroid carcinomas, potentially by acting as a negativeregulator of CTNNB1-dependent transcription in thesecancers61,62. Importantly, ablation of endogenous WNT5Ain mouse B cells induces the development of spontane-

ous B cell lymphomas and chronic myeloid leukaemias(CMLs)63, suggesting a tumour suppressor role forWNT5A in some cell types. However, WNT5A does notalways act as a growth and tumour suppressor. ReducingWNT5A expression in pancreatic cancer cells attenuatesxenograft tumour growth64, and WNT5A-transducedfeeder cells enhance the growth of patient-derived chroniclymphocytic leukaemia (CLL) cells65.

WNT receptors. Targeting WNT receptors that maintainmalignant phenotypes, but which are dispensable for nor-mal tissue homeostasis, may provide an attractive strategyfor therapeutic intervention in cancer. It is first necessary

to identify specific WNT receptor proteins that areexpressed in tumours and that are functionally relevantto disease progression. Several WNT receptors, includingFZD6 , are overexpressed in spontaneous B cell leukaemiasand lymphomas that occur in the T cell lymphoma break-point 1 (Tcl1+/−) mouse model. FZD6 has a unique rolein promoting leukaemia development in these animals,as Tcl1+/− Fzd6 −/− double-mutant mice are at significantlyless risk of leukaemia than Tcl1+/− Fzd6 +/+ littermates. By

contrast, leukaemogenesis is neither enhanced nor inhib-ited when Fzd9 is deleted66. FZD7 expression is increasedin many different human tumours compared with nor-mal tissues67 and also promotes cancer proliferation andprogression. Specifically, FZD7 knockdown reduces TCF-dependent transcription and xenograft tumour growth oftriple-negative breast cancer68 and inhibits the growth ofHCC cells69, which also rely on active WNT–CTNNB1signalling for their proliferation70.

In addition to FZD receptors, the WNT receptorsROR1 and ROR2 also contribute to cancer proliferationand tumorigenesis. Reducing ROR1 expression usingsmall interfering RNAs (siRNAs) decreases the growth

Figure 2 | CTNNB1-independent signalling pathways. Some WNTs, such as WNT5A and WNT11, fail to stabilizeβ-catenin (CTNNB1). Instead, they regulate signalling pathways that are associated with cell polarity and migration.

CTNNB1-independent WNT pathways are also initiated by the binding of certain WNTs, such as WNT11 and WNT5A, to

frizzled (FZD) receptors in order to activate Dishevelled (DVL), which can then activate a variety of downstream effectors.

In addition to regulating calcium-dependent and small-GTPase-dependent signalling networks, CTNNB1-independent

WNTs also regulate the planar cell polarity (PCP) signalling pathway. Disruption of FZD receptors also results in PCP

defects195–197, suggesting that a WNT–FZD signalling pathway may regulate PCP. In addition to FZD receptors, other

transmembrane proteins, such as VANGL, PTK7 and CELSR, genetically or biochemically interact with WNTs and FZDs to

regulate PCP signalling in vertebrates198. RYK and ROR receptor tyrosine kinases can also act as WNT receptors to activate

CTNNB1-independent signalling10. Like other WNT pathways, PCP also requires the intact function of DVL and numerous

other cytosolic factors, including SCRIB and PRICKLE198. In some contexts, CTNNB1-independent WNTs regulate small

GTPases, such as RHOA, RAC and CDC42, in a DVL-dependent manner199. WNT5A and WNT11 can also induce a calcium

flux, which results in the activation of various signalling pathways, such as protein kinase C (PKC), calcium/calmodulin-

dependent protein kinase II (CAMKII) and JUN N-terminal kinase (JNK)11. Pharmacological inhibitors are indicated in this

figure in blue boxes. AP-1, activator protein 1; ARHGEF7, RHO guanine nucleotide exchange factor 7; DAAM1,

Dishevelled-associated activator of morphogenesis 1; GIT, G protein-coupled receptor kinase-interactor 1; NFAT, nuclear

factor of activated T cells; PAK, p21-activated kinase; PLC, phospholipase C; SFRP, secreted frizzled-related protein;

WIF, WNT inhibitory factor.

R E V I E W S





of gastric, lung and breast cancer cells both in cell cultureand in xenografts71–73, and ROR2-targeted short hairpinRNAs (shRNAs) reduce the growth of leiomyosarcomaand renal cell carcinoma xenografts74,75. Deciphering thesignalling pathways acting downstream of ROR1 andROR2 remains an active area of research. In some can-cers, ROR1 supports tumorigenesis through crosstalkwith other RTKs, such as MET and epidermal growthfactor receptor (EGFR)71,73. In CLL cells, treatment withantisera against ROR1 blocks the enhanced prolifera-tion that is induced by WNT5A, thus suggesting thatROR1 might be mediating the mitogenic signal fromWNT5A65. Whether specific WNTs regulate ROR1- andROR2-driven tumorigenesis, and whether these WNTsalso regulate crosstalk between ROR receptors and otherRTKs, remain as open questions.

SFRPs and WIFs. WNT pathways are also regulated bya variety of secreted proteins, such as WNT inhibitoryfactors (WIFs) and secreted frizzled-related proteins(SFRPs) that can competitively displace certain WNTligands from their receptors. In some cancer models, anincrease in SFRP levels attenuates cancer growth, par-ticularly in cells that require autocrine WNT stimula-tion such as myeloma cells23 and subsets of breast cancercells76,77. Other cell types seem to be insensitive to alteredSFRP levels, and, in certain cancer contexts, SFRP canenhance cell growth. In contrast to the effects on otherbreast cancer cell lines76,77, SFRP1 is insufficient to inhibit

TCF-dependent transcription and has no effect on thexenograft growth of SUM1315 breast cancer cells78, sug-gesting cell-line-specific differences in SFRP sensitivity.SFRPs also regulate prostate cancer cell proliferation ina context-dependent manner, as the overexpression ofSFRP4 or SFRP3 decreases the proliferation of humanPC3 cells in vitro79,80, whereas the overexpression of SFRP1promotes the growth of BPH1 prostate cancer cells81.

WIF1 also regulates CTNNB1-dependent transcrip-tion and inhibits the proliferation of various cancer celllines, including those derived from cervical and prostatecancers, as well as glioblastoma82–84. Of note, overexpres-sion of WIF1 also inhibits osteosarcoma cell growth insoft agar assays and in xenograft assays, yet has littleeffect on CTNNB1-dependent transcription85. It is con-ceivable that WIFs and other secreted WNT antagonists

result in pleiotropic signalling outputs and regulate notonly CTNNB1 signalling, but potentially other signallingpathways also. SFRPs and WIFs associate with multi-ple WNTs, so altering SFRP and WIF levels could havepleiotropic effects on cancer cell proliferation.

WNTs and tumour-initiating cellsWNT signalling pathways contribute to both the main-tenance and differentiation of a variety of multipotentprogenitor cells in developing embryos and in adults.Numerous studies indicate that WNT–CTNNB1signalling analogously contributes to cancer progres-sion through the maintenance of highly tumorigenic

Table 1 | Somatic mutations in WNT pathway genes in cancers*

Gene Type of mutation Primary tissues Number ofmutated samples

% mutated Total samples

APC Primarily frameshift and deletion mutationsleading to compromised ability to degradeCTNNB1

Large intestine 2152 39% 5517

Stomach 129 15% 214

Soft tissue 50 12% 430

Small intestine 34 16% 214

Pancreas 26 14% 184

Liver 11 12% 94

CTNNB1 Mutations in CTNNB1 cluster aroundthe amino-terminus and prevent thephosphorylation of amino acids, S33, S37, T41and S45, resulting in impaired degradationof CTNNB1

Liver 907 23% 3933

Soft tissue 673 42% 1601

Endometrium 218 20% 1098

Kidney 168 14% 1225

Pancreas 125 26% 476

Ovary 104 11% 913

Adrenal gland 100 19% 534

Pituitary 86 24% 360

Biliary tract 43 10% 433

AXIN1 Many mutations prevent AXIN1 from actingas a scaffold to degrade CTNNB1

Biliary tract 10 38% 26

Liver 49 11% 448

WTX (also knownas FAM123B)

Predicted to be loss-of-function mutations Kidney 125 13% 949

Large intestine 19 13% 151

TCF7L2 Unknown Large intestine 13 28% 47

*Curated from the Catalogue of Somatic Mutations in Cancer (COSMIC) database. Genes that are mutated in at least 10% of the analysed samples for each cancertype are included in the table. APC, adenomatous polyposis coli; CTNNB1, β-catenin; WTX , Wilms tumour gene on the X chromosome.

R E V I E W S



http://www.sanger.ac.uk/genetics/CGP/cosmic






Basal-like breast cancers

A subset of breast cancers that

are characterized by a gene

expression signature similar to

that of the basal and

myoepithelial cells of the

breast.

subpopulations of cancer cells called tumour-initiating cells86.Perhaps the most extensive evidence for the significanceof WNT–CTNNB1 signalling in tumour-initiating cellscomes from studies of mouse- and patient-derived leu-kaemias. Subpopulations of leukaemia stem cells that arecapable of forming tumours with short latency in mice87,as well as myeloid progenitors isolated from patients withCML88, have increased levels of nuclear CTNNB1 and

increased WNT–CTNNB1 reporter activity. This sug-gests that WNT–CTNNB1 signalling is upregulated inleukaemia-initiating cells. Deletion of a floxed allele ofCtnnb1 inhibits the formation of various leukaemias inmice, including: the initiation of mixed-lineage leukaemia(MLL) driven by the Mll–Enlfusion oncogene87, the initia-tion of CML driven by the Bcr–Abl1 fusion oncogene89 andthe progression of acute myeloid leukaemia (AML) driven

Table 2 | WNT signalling proteins are associated with distinct patient outcomes in a cancer-subtype-specific manner*

Protein Cancer Clinical relevance

APC Breast APC expression was decreased in grade 1 breast tumours compared with normal breast, yet was increased in grade 3tumours

CTNNB1 Adrenocortical High nuclear CTNNB1 levels were associated with reduced overall and disease-free survival

AML CTNNB1 is expressed in a subset of primary AML samples and correlates with decreased relapse-free and overall survival

Breast Nuclear CTNNB1 is associated with reduced metastasis-free and overall survival in breast cancer. Cancer subtypedifferences have been observed. Invasive ductal carcinomas exhibited membranous but not nuclear CTNNB1 staining,lobular carcinomas lacked any CTNNB1 expression, and basal-like breast cancers exhibited strong nuclear CTNNB1levels and high expression of CTNNB1 target genes

Colorectal High nuclear CTNNB1 expression was associated with patient deaths, particularly when found at the invasive front oftumours. However, CTNNB1 levels were not prognostic in obese patients

Oesophagealcarcinoma

Nuclear CTNNB1 was increased in malignant tumours and correlates with poor one-year survival but not with lymphnode metastases

Gastric cancer Decreased nuclear CTNNB1 expression was observed in high-grade gastric cancers

Glioblastoma High nuclear and cytoplasmic CTNNB1 levels were associated with poor survival in glioblastoma

Lung Cytoplasmic or nuclear CTNNB1 expression predicts increased patient survival in non-small-cell lung carcinomas andnon-squamous-cell lung carcinomas

Prostate Nuclear CTNNB1 correlates with decreased relapse-free survival, and nuclear CTNNB1 was decreased in metastases

Melanoma High levels of nuclear CTNNB1 in primary tumours predicted patient deaths

HCC High levels of CTNNB1 were predictive of decreased overall survival and increased risk of recurrence

TCF/LEFfamily

Colorectal High LEF1 but low TCF4 expression were correlated with a better prognosis in colorectal carcinoma. However, inanother study, high levels of LEF1 were associated with reduced patient survival in colorectal cancer, and the reasons forthis discrepancy are unclear

ALL High LEF1 transcript expression was associated with poor relapse-free survival in B cell ALL

ROR1andROR2

Breast ROR1 expression was increased in high-grade and triple-negative breast cancer, and high levels of ROR1 correlatedwith decreased patient survival

Sarcoma ROR2 is expressed in a subset of soft tissue sarcomas, including leiomyosarcoma and gastrointestinal stromal tumours,and predicts patient deaths

SFRP4 Prostate Decreased membranous expression of SFRP4 was associated with decreased patient survival in prostate cancer

WNT1 Breast WNT1 protein expression was increased in tumour tissue compared with non-cancerous adjacent tissue. However,

there was no significant difference in WNT1 expression in high-grade breast tumours, indicating that WNT1 may beparticularly important during the early stages of tumorigenesis

Glioblastoma Increased WNT1 staining was associated with poor survival in glioma

WNT2 Oesophageal Overexpression of WNT2 was correlated with poor survival in oesophageal squamous cell carcinoma. WNT2 was highlyoverexpressed in tumour-associated fibroblasts, indicating that it might function non-cell-autonomously

WNT5A Gastric Increased levels of WNT5A protein were associated with high-grade tumours and with decreased patient survival

Prostate Low WNT5A, especially in combination with high Ki67 staining or high androgen receptor protein levels, was predictiveof reduced relapse-free survival

Ovarian High WNT5A was correlated with poor survival in ovarian carcinoma, yet low expression of WNT5A is observed duringprogression in the epithelial subtype of ovarian cancer, and loss of WNT5A correlates with decreased overall survival inpatients with epithelial ovarian cancer

Colorectal High levels of WNT5A protein were correlated with increased patient survival in locally invasive colon cancer

WNT7A Ovarian WNT7A was overexpressed in ovarian carcinoma and was significantly associated with patient deaths

WIF1 HCC Loss of WIF1 mRNA expression was associated with decreased overall survival in HCC

*A full table with references can be found in Supplementary information S1 (table). ALL, acute lymphoblastic leukaemia; AML, acute myeloid leukaemia; APC,adenomatous polyposis coli; CTNNB1, β-catenin; HCC, hepatocellular carcinoma; ROR, receptor tyrosine kinase-like orphan receptor; SFRP4, secretedfrizzled-related protein 4; WIF1, WNT inhibitory factor 1.

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Tumour-initiating cells

A subset of cancer cells, often

with stem-cell-like expression

profiles, that are capable of

generating new tumours.

Mouse mammary tumour

virus

(MMTV). A species of retrovirus

that can drive mammary

adenocarcinoma development

in susceptible strains of mice in

the presence of steroid

hormones.

by the Hoxa9 and Meis1a oncogenes90. Loss of CTNNB1

also

reduces the tumour-forming ability of serially trans-planted CLL cells but not acute lymphoblastic leukaemia(ALL) cells, thus suggesting different requirements forCTNNB1 in leukaemia subtypes89. CTNNB1-dependenttranscription was low in the ALL cells but high in the CLLcells89. The authors of this study suggest that activationof CTNNB1-dependent transcription is required for theinitiation of forms of leukaemia that are characterized bya progenitor cell hierarchy.

CTNNB1-dependent transcription in colon pro-genitor cells similarly drives the initiation of colorectaltumours. Deletion of Apc in colon crypt progenitor cellsinduces rapid and sustained adenoma growth in mice,whereas deletion of Apc in non-progenitor cells doesnot induce sustained tumours91. Increased CTNNB1levels in colon and intestinal tissue drives tumori-genesis through the activation of a specific subset of theTCF/LEF family in order to promote a progenitor-likegene expression signature92. Specifically, TCF7L2 andCTNNB1 regulate the expression of many target genesthat are normally associated with proliferative progeni-tor cells of colon crypts92. Transcription cofactors suchas BCL9 and BCL9L enhance the expression of a sub-set of CTNNB1 target genes that are associated withprogenitor cell phenotypes in the colon93. These geneexpression changes are probably functionally relevantbecause ablation of both BCL9 and BCL9L prevents

the generation of chemically induced colorectal adeno-mas in mice93. BCL9L protein levels were also recentlyfound to be upregulated in patients with ovarian car-cinoma that persisted after surgery 94, suggesting thatthese CTNNB1 transcription cofactors might promotetumorigenic properties of multiple carcinomas.

WNT–CTNNB1 signalling also supports the self-renewal of both normal and malignant mammary stemcells. Cells expressing mammary stem cell markers areenriched in both premalignant mammary glands andspontaneous tumours in transgenic mice that expressWnt1 under the control of the mouse mammary tumour

virus ( MMTV ) promoter, but not when MMTV is used

to drive the expression of Hras, Erbb2 (also known

as Neu and Her2), or Polyoma middle T antigen(PyMT )95,96. This indicates that WNT–CTNNB1tumours contain progenitor-like cells. WNT path-way genes such as FZD6 and WNT7B are also highlyexpressed in undifferentiated mouse mammarytumour cells that are grown in spheroid conditions toenrich for progenitor-like cells, compared with differ-entiated mouse mammary cells97. Importantly, WNT–CTNNB1 is not only active, but is functionally relevantin mammary stem cells because WNT3A is sufficientto promote the self-renewal of mouse mammary stemcells grown as spheres in vitro and enhances the abil-ity of these mammary progenitor cells to reconstitute mammary glands in mice97,98.

Human mammary cells expressing progenitor cell sur-face markers similarly express altered levels of many WNTsand WNT modulators. For example, relative to non-tumorigenic mammary cells, a subpopulation of mes-enchymal human mammary cells that induce xenografttumours at high efficiency have reduced expression ofsecreted inhibitors of WNT–CTNNB1 signalling, such asSFRP1 and dickkopf 1 homologue (DKK1), but increasedexpression of WNT5A99. Restoration of the expressionof SFRP1 but not DKK1 in human mesenchymal mam-mary cells reduces secondary sphere formation in vitro and tumorigenesis in xenograft models99. Further researchis necessary to determine why SFRP1 inhibits mammary

tumorigenesis by mammary progenitor-like cells, whereasDKK1 has no effect99. Together, these studies indicatethat WNT signalling pathways can influence progenitorpopulations in a manner that affects tumorigenesis.

WNT signalling and apoptosis and senescence

WNT signalling pathways not only drive the initiation oftumorigenesis, but are also required as tumours continueto grow. In some cell types, WNT stimulation prevents cel-lular senescence. For example, loss of endogenous WNT2in fibroblasts leads to increased expression of senescencemarkers, whereas stimulation with WNT3A or inhibi-tion of glycogen synthase kinase 3β (GSK3β), which is a

Box 1 | Context-dependent transcriptional outputs of the TCF/LEF family and CTNNB1

Activating or inhibiting the function of different WNT pathway proteins can induce unique transcriptional responses in a

cell‑type‑dependent manner. For example, activating mutations in β‑catenin (CTNNB1) and loss‑of‑function mutations in

AXIN1 induce different patterns of gene expression in hepatocellular carcinoma (HCC)183. These different transcriptional

responses could arise from different levels of WNT–CTNNB1 pathway activation given that AXIN1 mutations induced

much lower levels of CTNNB1‑dependent reporter activity than CTNNB1 mutations183, but may also involve

CTNNB1‑independent functions of AXIN1. Furthermore, disruption of specific TCF/LEF family members (mammals

express four different TCF/LEF family transcription factors with unique roles in regulating WNT target genetranscription184) is not equivalent to the ablation of CTNNB1. This is revealed by expression‑profiling studies indicating

little overlap between gene expression changes induced by small interfering RNAs (siRNAs) targeting CTNNB1 versus

TCF7L2185. Although a dominant‑negative form of TCF7L2 reduced CTNNB1‑dependent transcription and induced cell

cycle arrest in colorectal cells92,185, increasing or decreasing TCF7L2 expression either activated or repressed

CTNNB1‑dependent reporters in other cell lines185. Cell‑type‑specific WNT–CTNNB1 transcriptional programs may arise,

in part, from direct interactions between CTNNB1 and other transcription factors. For example, several studies also

indicate that CTNNB1 can interact with nuclear hormone receptors, such as the androgen receptor186, the vitamin D

receptor187 and the retinoic acid receptor188. CTNNB1 can regulate the transcriptional activity of these receptors and vice

versa. Overexpression of CTNNB1, or treatment with WNT3A, can regulate androgen‑receptor‑dependent

transcription186,189, whereas activation of the vitamin D receptor inhibits CTNNB1‑dependent transcription in colorectal

cancer cells187,190.

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Scratch assays

Assays that are used to

determine the motility of cells

in vitro. In these experiments,

cell monolayers are scratched

or wounded and the ability of

cells to migrate and fill the

resulting gap is measured.

negative regulator of WNT–CTNNB1 signalling (FIG. 1),delays both replicative and oncogene-induced senes-cence100. Ectopic expression of WNT1 in mouse mam-mary cells similarly prevents contact-inhibition-inducedsenescence and supports the development of three-dimensional outgrowths from cell monolayers98. OtherWNTs promote rather than prevent senescence. WNT5Alevels increase as late passage primary ovarian epithelialcells begin to senesce, and the overexpression of WNT5Ain ovarian epithelial carcinoma cells leads to senescenceboth in vitro and in xenografted tumours61. Many differ-ent cancer cell types require endogenous WNT stimula-tion for survival when challenged with chemotherapeuticagents or other toxins101–109. Finally, identifying WNTreceptors that are necessary for cancer cell evasion ofsenescence and apoptosis could inform clinical strate-gies. Many studies indicate an important role for ROR1in cancer cell survival, as ROR1-targeted siRNAs induceapoptosis in CLL, breast carcinoma and cervical carci-noma cells72,110,111. Blocking FZD7 activity using siRNAsor peptides can similarly induce apoptosis in colon and

breast cancer cells67,68,112,113.

WNT signalling in metastatic disease

CTNNB1-dependent signalling and metastasis. WNT–CTNNB1 signalling also contributes to the metastaticprogression of cancer, and can either enhance or inhibitmigratory behaviour in a cancer cell-type-specific man-ner. Both WNT1 and WNT3A promote the migrationof myeloma cells isolated from patients114, whereas adominant-negative form of LRP5 can negatively regu-late WNT–CTNNB1 signalling and abrogates the inva-sion and migration of prostate cancer cells80. Consistentwith these observations indicating that WNT–CTNNB1signalling enhances cell migration in some cancers,many secreted antagonists of the WNT–CTNNB1pathway (such as DKK1 and SFRPs) slow cancer cellmotility and block invasive behaviour,44,80,99,115. Otherstudies suggest that WNT–FZD signalling can alsoinhibit cancer cell migration and invasion. For exam-ple, ectopic expression of FZD1 decreases the invasionof both primary thyroid cells and thyroid carcinomacells through Matrigel116, and the addition of mediafrom cells expressing WNT2 slows the migration andinvasion of oesophageal carcinoma cells117. What arethe mechanisms by which WNT–CTNNB1 signal-ling regulates malignant cell migration and inva-sion? In some cases hyper- or hypo-activation

of the WNT–CTNNB1 pathway can regulate cellmigration through the differential expression ofCTNNB1 target genes. WNT1 overexpressioninduces the invasiveness of MCF7 breast cancercells , and this effect can be attenuated by blockingCTNNB1-dependent transcription118. Activation ofWNT–CTNNB1 signalling in cancer often drivesa transcriptional program that is reminiscent of anepithelial–mesenchymal transition (EMT)119, whichcan promote cell migration and invasiveness (BOX 2).In addition to promoting EMT-like changes in a sub-set of cancers, CTNNB1 also regulates the expres-sion of other factors that are relevant to metastatic

progression, notably matrix metalloproteinases(MMPs) and other factors that are necessary for theregulation of the extracellular matrix10.

Components of the WNT–CTNNB1 pathway alsodirectly participate in coordinating changes in cell mor-phology, polarity and signalling that are necessary formigration and invasion. Core WNT signalling path-way components localize to specific subcellular regionsin migrating cells and contribute to the establishmentof cell polarity as these cells migrate. APC is recruitedto the leading edge of astrocytes migrating in wound-healing assays where it promotes the localization ofdiscs large homologue 1 (DLG1)120, which is anotherimportant polarity protein. Dishevelled (DVL) andAXIN are also required for the establishment of orga-nelle polarity of rat embryonic fibroblasts migrating inscratch assays, whereas GSK3β inhibitors, which acti-

vate CTNNB1-dependent transcription, similarly prevent organelle polarity in migrating fibroblasts121. Theseobservations suggest that tight control of pathway activa-tion is necessary for cell migration.

Relatively few studies have addressed potential rolesfor WNT signalling in animal models of metastasis . Expression of dominant-negative TCF1 and TCF4inhibits the metastatic capabilities of lung adeno-carcinoma cells in a mouse model122, whereas blockingCTNNB1 function using shRNAs reduces the incidenceof both pulmonary and abdominal metastatic lesionsin xenograft models of HCC123. Although CTNNB1inhibits melanoma cell migration in vitro124,125, expres-sion of stabilized, constitutively active CTNNB1 coop-erates with PTEN loss and NRAS overexpression topromote metastatic progression in mouse models ofmelanoma126,125. One possible explanation for this dis-crepancy is that WNT–CTNNB1 signalling mediatescomplex cell–cell interactions that are necessary formetastatic progression in animal models. For example,one recent study found that metastatic breast cancercells induce the expression of periostin (POSTN) in thelung microenvironment, which can, in turn, enhanceWNT–CTNNB1 signalling and the growth of metastaticlesions127.

CTNNB1-independent WNT pathways and metastasis.

During development, CTNNB1-independent WNTsignalling pathways have crucial roles in embryo mor-phogenesis, in part, through the regulation of directedcell migration. These pathways may analogously con-

tribute to the migration and invasion of cancer cells ina context-dependent manner. In melanoma, WNT5Aacts through CTNNB1-independent signallingmechanisms to enhance melanoma cell invasivenessin vitro42. However, in other cancer cell lines, WNT5Ainhibits cell migration and invasiveness. For example,recombinant WNT5A reduces the invasion of 22Rv1and DU145 prostate cancer cells128, and inhibits themigration of multiple breast cancer cell types129,200.

Studies investigating the mechanisms by whichWNTs can regulate cell migration and metastasis inde-pendently of CTNNB1 are still in their infancy. Severalstudies have suggested that altering WNT5A signalling

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Planar cell polarity

(PCP). The collective

orientation of cells within the

epithelial plane.

may induce changes in cell polarity. Supporting thisidea, expression of WNT5A enhances the polarizationof melanoma cell adhesion molecule (MCAM) in mela-noma cells that are responding to a chemokine gradi-ent130. Moreover, knockdown of endogenous WNT5Ausing siRNAs similarly disrupts organelle polarity inrat embryonic fibroblasts migrating in scratch assays121.CTNNB1-independent WNTs may also regulate cellpolarity in migrating cancer cells by cooperating withother planar cell polarity (PCP) proteins, such as SCRIB,VANGL and PTK7 (REFS 131–135). In developingembryos, WNT5A regulates the phosphorylation andlocalization of VANGL136, which suggests that WNTscan either directly or indirectly modulate the activityof PCP proteins. Further research is necessary to fullyunderstand the crosstalk between WNT–FZD signallingand PCP signalling networks in metastatic cancer cells,especially in vivo.

In addition to either directly or indirectly regulat-ing cell polarity, CTNNB1-independent WNTs canalso regulate cell motility and invasiveness by activat-ing kinase-dependent signalling cascades, as is evidentfrom the observations that WNT5A-induced inva-

sion of MCF7 cells can be inhibited by blocking JNKsignalling115 and that WNT5A-induced invasivenessof melanoma requires the phosphorylation of pro-tein kinase C (PKC)42. Although many studies foundthat WNT3A can activate CTNNB1-dependent sig-nalling, WNT3A-induced migration of myeloma cellscan be blocked by PKC inhibition, but not by DKK1(REF. 114). In a previous study, DKK1 expression was suf-ficient to block WNT3A-induced CTNNB1-dependenttranscription50, suggesting that WNT3A can regulatemyeloma cell migration independently of CTNNB1.Finally, recent studies highlight an important role forthe WNT5A receptor ROR2 in mediating metastatic cell

behaviour. siRNA-mediated knockdown of ROR2 in B16mouse melanoma cells reduces the frequency and sever-ity of lung metastases in mice137. ROR2-targeted siRNAalso inhibits both baseline and WNT5A-induced inva-sion of sarcoma cells in vitro75,138 , whereas ROR2 over-expression enhances leiomyosarcoma and osteosarcomacell migration75,138. Although the mechanisms are notentirely clear, these studies indicate that ROR2 mayenhance migration by promoting the expression ofMMPs75,138.

Context-dependent WNT signalling in cancer

Unique outcomes of specific WNT pathway aberrations. WNT pathway genes are mutated at different frequen-cies in different cancer subtypes, suggesting that thesemutations do not equivalently activate WNT signallingand may have unique functional outcomes. For exam-ple, APC mutations are common in colorectal tumoursbut are rare in HCC15 and in melanoma139. Mutationsin APC can present unique phenotypes partially owingto the multiplicity of APC functions as a component

of the destruction complex in the WNT pathway, asa microtubule-binding protein and as a guardian ofgenome integrity 140.

It is also likely that the activation of WNT signal-ling pathways by different means results in differencesin signalling amplitude or duration, which mightlead to unique physiological or pathological conse-quences. Expression of a stabilized, constitutivelyactive form of CTNNB1 driven by the MMTV pro-moter is sufficient to induce extensive lobuloalveolardevelopment and adenocarcinomas in mouse mam-mary glands but does not induce ductal branching 141.By contrast, expression of WNT1 induces extensiveductal branching and mammary tumours with longlatency 4,141,142. These results suggest that WNT1 andconstitutively active CTNNB1 promote different phe-notypes in mouse mammary tissue. Interestingly, MMTV–Wnt1 induces CTNNB1-dependent tran-scription in basally located mammary cells, whereas MMTV–Ctnnb1 drives CTNNB1-dependent tran-scription in mammary cells expressing luminal mark-ers142. These data suggest that different subsets of cellsin mouse mammary tissue vary in their responsive-ness to different means of activating WNT–CTNNB1signalling. Surprisingly, the overexpression of a con-stitutively active form of CTNNB1 in other secretoryepithelia induces tumours in a tissue-specific man-

ner: CTNNB1 induces neoplastic growth of the pros-tate, but promotes cell hyperplasia and keratinizationof the salivary glands and skin143.

Crosstalk with other oncogenes and tumour suppressors. Aberrations in WNT signalling pathways drive tumori-genesis in cooperation with other signalling pathways,oncogenes and tumour suppressors. Although a com-prehensive review of crosstalk between WNT signallingand other signalling pathways is beyond the scope of thisReview, below we discuss a few instances of crosstalkbetween WNT–CTNNB1 signalling and key tumoursuppressors and oncogenes.

Box 2 | WNT signalling and the epithelial–mesenchymal transition

The development of metastases is thought to involve the delamination of cells from

primary tumours through an epithelial–mesenchymal transition (EMT). The process of

EMT involves alterations in protein expression resulting in complex changes in cell

behaviours, such as reduced cell–cell adhesion and enhanced motility119,191. Numerous

studies indicate that the activation of WNT–β‑catenin (WNT–CTNNB1) signalling can

promote transcriptional changes in order to drive EMT in cancer. Expression of either

WNT1, stabilized CTNNB1, or AXIN2 (which is a WNT–CTNNB1 target) is sufficient toinduce EMT‑like changes in MCF7 breast cancer cells, including upregulation of SNAI1

and downregulation of E‑cadherin expression118. Other WNT signalling molecules seem

to suppress cell migration and invasiveness by inhibiting or reversing EMT. For example,

the overexpression of WNT5A or the addition of recombinant WNT5A increases the

expression of CTNNB1 and E‑cadherin at the plasma membrane in breast cancer

cells192, whereas the expression of dickkopf 1 homologue (DKK1) inhibits EMT in

colorectal cancer193. Overexpression of either SFRP3 or a dominant‑negative form of

LRP5 that lacks the cytosolic and extracellular domains similarly induced the

expression of E‑cadherin and reduced the expression of N‑cadherin, SLUG and TWIST

in prostate cancer80. Connections between WNT signalling and EMT are further

complicated by studies indicating that members of the WNT pathway are also

upregulated in response to EMT. For example, WNT signalling proteins such as ROR2

and WNT5A

were upregulated following SNAI1‑induced EMT in epidermal carcinoma

cells194. In renal cell carcinoma, ROR2 expression seemed to promote EMT‑like

changes74, thus raising the possibility that positive feedback loops between EMTprograms and ROR2‑dependent signalling could be important in some cancers.

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Apcmin/min mice

Mice that are homozygous for

the Apcmin allele. Apcmin is a

mutant form of Apc that leads

to familial adenomatous

polyposis (FAP) and

spontaneous colorectal

tumours.

Altered activity of WNT signalling pathways is notsufficient to drive tumorigenesis. This is evident fromstudies revealing that the expression of a constitutivelyactive form of CTNNB1 simultaneously enhancesproliferation and increases apoptosis in mouse intes-tinal tissue144. Overexpression of constitutively activeCTNNB1 in mouse embryonic fibroblasts (MEFs) simi-larly induces a senescence-like phenotype rather thanenhanced proliferation145. However, CTNNB1-inducedsenescence is not observed when constitutively activeCTNNB1 is expressed in p53-deficient MEFs, or in ARF-deficient MEFs (which lack the ARF-encoding exon ofCdkn2a)145. Inactivation of p53 also has an importantrole in mouse models of WNT–CTNNB1-inducedtumorigenesis. Apcmin/min mice with a Trp53−/− backgrounddeveloped more intestinal adenomas than Apcmin/min micewith a Trp53+/+ background146. These results suggest thatthe apoptosis or senescence that is observed in responseto hyperactivation of WNT–CTNNB1 signalling arisesat least in part by p53 signalling.

Research using mouse models of other cancers

has similarly reported nodes of crosstalk betweenWNT signalling networks and various oncogenes. Inone model of lung cancer, overexpression of consti-tutively active CTNNB1 produces no lung tumours,whereas co-expression of constitutively active formsof both KRAS and CTNNB1 induces lung tumours147.Expression of constitutively active CTNNB1 also syn-ergizes with active forms of NRAS to induce melano-mas with short latency 148. Similarly, expression of eitherthe human papillomavirus 16 (HPV16) E7 viral onco-protein or constitutively active CTNNB1 promotes thedevelopment of cervical tumours with long latency,whereas mice co-expressing these proteins developedcervical pathologies at a much higher rate149. Finally,co-transfection of the hepatitis C virus (HCV) core pro-tein with CTNNB1 synergistically enhances the growthof xenografted HCC cells70. Together, these studies (andadditional findings not discussed here owing to spacelimitations) indicate that aberrant CTNNB1 signallingcan cooperate with various other oncogenes to promotethe development of aggressive carcinomas.

Targeting WNT signalling in cancerSmall molecules. A few US Food and Drug Administration(FDA)-approved drugs modulate WNT signallingin vivo, but these drugs also have other cellular targets.For example, lithium chloride has been in clinical use

for decades and activates CTNNB1 by inhibiting GSK3.Non-steroidal anti-inflammatory drugs (NSAIDs) andthe selective COX2 inhibitor celecoxib can inhibitCTNNB1-dependent transcription in colorectal cells150–152 and reduce polyp formation in patients with FAP and inmouse models of colon cancer 153–156. This suggests thatthese drugs may act in part through the modulation ofCTNNB1 signalling. Numerous studies have used high-throughput screening of WNT-activated luciferase report-ers to identify novel inhibitors of WNT signalling. Forexample, the drug IWP (‘inhibitor of WNT production’)was identified in a screen of a synthetic chemical libraryand was found to inhibit the activity of Porcupine; this is

a membrane-bound acetyltransferase that modifies WNTligands with a palmitoyl group that is required for theirsecretion and signalling activity 157. Two recent studieshave identified the small molecules XAV939 and pyrvin-ium using reporter-based screening approaches. XAV939enhances AXIN stability through tankyrase inhibition,whereas pyrvinium promotes CTNNB1 phosphorylationthrough casein kinase activation158,159.

As the activation and inactivation of WNT signallingpathways through mutations and differential expressionof WNT pathway proteins can lead to unique responsesin different cancers, the identification of small mol-ecules targeting specific WNT signalling componentsmay be therapeutically useful. Hyperactivation of theWNT–CTNNB1 pathway due to mutations in APCand AXIN1 limits the potential molecular targets forpathway modulation because factors acting upstreamof the destruction complex are no longer necessary forpathway activation. To overcome these challenges sev-eral researchers have conducted screens to identify mol-ecules that can disrupt the interaction between TCF7L2

and CTNNB1 and thus inhibit CTNNB1-dependenttranscription97,160–163. Several of the identified com-pounds inhibited the growth of colorectal carcinomacells in vitro and in vivo160–162, and additional compoundssimilarly prevented the initiation and growth of mam-mary tumours in mouse xenografts97. The developmentof specific inhibitors of DVL using protein–proteininteraction screens and structure-based design algo-rithms has also been attempted with some success,and a few of these small molecules have been shownto regulate WNT–CTNNB1 signalling164–168. It shouldbe noted that these DVL inhibitors could theoreticallyinhibit DVL function in both CTNNB1-dependent andCTNNB1-independent pathways.

Thus far, direct targeting of WNT signalling hasbeen difficult largely owing to the lack of pathway-specific targets and the potential redundancy ofmany pathway components. Despite these challenges,numerous small-molecule inhibitors of WNT signal-ling pathways have been identified, as described above.At present, far fewer published studies describe eitherWNT signalling activators or synergists. It is unclearwhether the scarcity of effective WNT-activatingcompounds reflects the biology of WNT signalling,or rather reflects a reluctance of researchers to focuson developing such compounds given the longstand-ing concerns about the oncogenic effects of hyperac-

tivated WNT signalling in some cancers. For a morecomprehensive overview of small molecules targetingWNT signalling pathways and their molecular targetsplease refer to TABLE 3, Supplementary information S2 (table) and FIGS 1,2.

Blocking antibodies. WNT–CTNNB1 signalling isrequired for serial transplantation and self-renewal ofboth normal haematopoietic stem cells and subsetsof leukaemia stem cells88–90,169,170. This suggests that gen-eral inhibition of CTNNB1 signalling in patients couldhave unwanted side effects on normal, adult stem cells.Instead, targeting specific WNTs and WNT receptors

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that are aberrantly overexpressed in tumours may proveto be an attractive strategy for targeting WNT signal-ling pathways preferentially in cancer cells. Indeed, sev-eral WNT-blocking antibodies inhibit proliferation andinduce apoptosis in different cancers109,171–172. Promisingresults have also been obtained using WNT-blockingantibodies in vivo. For example, intraperitoneal injec-tions of WNT3A-neutralizing antibodies decrease pro-liferation and induce apoptosis in a mouse model ofprostate cancer53.

Other studies have used blocking antibodies tar-geting WNT receptors to inhibit cell growth and toinduce apoptosis in cancer cells. These agents includeFZD7-specific antibodies, which preferentially tar-get colon cancer and HCC cells as opposed to normaltissue69,173. Similarly, LRP-targeted antibodies reducethe growth of allografted tumour cells derived from MMTV–Wnt1 and MMTV–Wnt3 tumours174. It mayalso be possible to target WNT receptors that lack clearfunctional relevance to disease progression, as long asthose receptors are preferentially expressed in tumours

relative to normal tissue. In a mouse synovial sarcomaxenograft model, FZD10-targeted antibodies havebeen used to deliver the radioisotope Yttrium-90 totumours, resulting in inhibition of tumour growth167.As ROR1 is highly expressed in embryonic tissues andblood cancers, but not in normal adult cells65, a similarapproach might be useful for targeting these cancers.Several groups are currently developing ROR1-targetedantibody derivatives or other techniques to target theROR1-positive malignant cells for selective killing bythe immune system111,175,176.

Peptides. In addition to WNT and WNT receptorantibody-mediated therapies, a few studies have sug-gested the utility of WNT-modulatory peptides.Supporting this approach, SFRP1 or SFRP1-derivedpeptides delayed HCT116 xenograft tumour formationin nude mice and reduced the proportion of mitotic,but not apoptotic, cells in these tumours177. Similarly,injection of full-length SFRP1 protein in xenograftedmammary tumours derived from transformed mes-enchymal precursor cells reduced tumour growth99.Another study identified peptide ligands that bind to thePDZ domain of human FZD2 and used this approachto disrupt WNT–CTNNB1 signalling in cell lines167.Peptides may also prove to be effective for targetingCTNNB1-independent signalling; however, the thera-

peutic potential of such approaches might be limitedgiven that the activity of many downstream kinases suchas PKC and JNK are important for normal homeostasisand metabolism. Full-length WNT5A and a formylatedhexapeptide of WNT5A inhibit the migration of bothHB2 normal mammary cells and MDA-MB-268 breastcancer cells in a transwell assay 129, which suggests thatpeptide mimetics of WNTs or other extracellular WNTpathway members might serve as a therapeutic strategy.Indeed, daily injections with a WNT5A-derived pep-tide called Foxy-5 reduced the number of lung and livermetastases formed in mice that were allografted with4T1 breast cancer cells178.

Numerous groups have attempted to generate pep-tides that can block high levels of FZD receptor activity,with some success. FZD7 extracellular domain peptidescan block TCF/LEF reporter activity and the expressionof WNT–CTNNB1 target genes in HCC cells69. TheseFZD7-blocking peptides can also interfere with thegrowth of HCC cells, which express high levels ofthe FZD7 receptor, but do not interfere with the viabil-ity of normal hepatocytes lacking FZD7 expression69,168.FZD7 peptides derived from the domains that interactwith DVL similarly inhibited the growth of HCC cells168;however, it is not clear whether this peptide specificallyblocks FZD7 function or perhaps more generally blocksDVL function in these cells. Like FZD7 extracellulardomain peptides, a soluble WNT receptor consistingof an FZD8 cysteine-rich domain fused to a human Fcdomain exhibits activity against teratoma lines in vivo179.A crystal structure of Xenopus laevis WNT8 bound to anFZD8 extracellular domain has recently been solved180,and this breakthrough may facilitate the design of futuremodulators of WNT signalling in cancer.

WNT signalling and combination therapy. Given thatderegulation of WNT signalling pathways is not suf-ficient to induce tumour formation, it is possible thatinhibition or activation of WNT signalling pathwaysalone will be insufficient to curb cancer progression. In addition to regulating the normal proliferation andsurvival of cancer cells in a context-dependent manner,activation or inhibition of WNT signalling pathways canalso either sensitize or desensitize cancer cells to toxicinsults, which might be advantageous in the develop-ment of combination therapies. Supporting this idea,inhibition of the WNT–CTNNB1 pathway in a vari-ety of cancer cells increases cell sensitivity to chemo-therapeutic agents. For example, WIF1 increases PC3prostate cancer cell sensitivity to paclitaxel and etopo-side, but has no effect on DU145 cell death181. DKK1similarly increases the sensitivity of U87MG glioblas-toma cells to various toxins including DNA-damagingagents and drugs targeting microtubules106. Conversely,increased activity of certain WNTs may sensitize can-cer cells to chemotherapeutic agents. Overexpression ofWNT5A sensitizes SKOV3 ovarian carcinoma cells to

various chemotherapeutic agents182, and activation ofWNT–CTNNB1 signalling can sensitize melanoma cellsto inhibitors of BRAF–MAPK signalling56. Collectively,these studies indicate that activating or inhibiting WNT

pathways in conjunction with more conventional chem-otherapeutics might result in cooperative inhibition oftumour growth.

Conclusions

Based on early discoveries linking the activation WNT–CTNNB1 signalling to breast and colon carcinomas, it hasgenerally been assumed that elevation of WNT signallingpromotes tumour initiation and progression. Subsequentstudies have suggested that this initial assumption mayan oversimplification. Instead, it seems that WNT–CTNNB1 signalling, as well as CTNNB1-independentWNT signalling pathways, can either promote or inhibit

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Table 3 | Modulators of WNT signalling and their effects on cancers*

Class of compoundand target

Compound Functional effects of drug in cancer model or in vivo

Inhibitor

PORCN(O-acetyltransferase),WNT protein production

IWP IWP did not regulate the proliferation of several different cancer cell lines in vitro

Stimulates CK1α topromote CTNNB1degradation

Pyrvinium Pyrvinium synergizes with 5-fluorouracil in mediating the apoptosis of SW620colorectal cancer cells and inhibits the proliferation of SW480 and HCT116 cells. Itshould be noted that pyrvinium is not a specific inhibitor of WNT signalling

Tankyrase inhibitor thatcan stabilize AXIN

JW55 Reduces tumour growth induced by LGR5 in an APC-mutant mouse model oftumorigenesis

IWR Inhibits tailfin regeneration in zebrafish, which is a WNT-dependent process

XAV939 Inhibits colony formation in soft agar of DLD1 colorectal cancer cells in anAXIN-dependent manner, but does not inhibit colony formation of RKO colorectalcancer cells in soft agar

CTNNB1 interactionwith CBP

ICG-001 Reduces polyp formation in Apcmin mouse model and decreases xenograft growth ofSW620 colon carcinoma cells

CTNNB1 interactionwith TCF7L2

iCRT3, iCRT5 and iCRT14 iCRT3 reduced the growth of colorectal cancer cells derived from patient biopsy samples;iCRT3, iCRT5 and iCRT14 inhibit WNT3A-dependent changes in mouse mammary cellmorphology, which have been previously associated with cellular transformation

BC21 Not reported

NC043 (15-oxospiramilactone) Inhibits SW480 cell tumorigenesis in a xenograft model and inhibits Caco-2 andSW480 colorectal cancer growth in vitro

PKF115-584, CGP049090 andPKF118-310

PKF115-584, CGP049090 and PKF118-310 can inhibit the growth of HCC cells inxenografts and axis duplication in frogs

CTNNB1 stability Thiazolidinediones (Δ2TG andSTG28)

Not reported

Murrayafoline A Reduces the viability of DLD1, SW480, HCT116 and LS174T colorectal cancer cells

CTNNB1- andTCF-dependenttranscription

OSU03012 Inhibits the growth of various medulloblastoma cell lines

3,6-dihydroxyflavone Inhibits the proliferation of MDA-MB-231 breast cancer cells

CCT036477, CCT070535 andCCT031374

Inhibits the growth of SW480 and HCT116 colorectal cell lines

Non-steroidal anti-inflammatory

drugs

Reduces polyp formation in FAP patients and tumour growth in mouse models of

colorectal cancer

DVL–FZD interaction NSC668036 Partially inhibits axis duplication in frogs induced by WNT3A but not by CTNNB1

3289-8625 Inhibits the growth of PC3 prostate cancer cells, yet it is unclear if this effect is due to aloss of WNT–CTNNB1 signalling

FJ9 Reduces the growth of H460 lung cancer cells in xenograft studies

PEN-N3 Not reported

Reduced DVL2 andCTNNB1 protein levels

Niclosamide Inhibits the growth of HT29, HCT116 and Caco-2 colorectal cells in culture and reducesthe growth of colorectal cancer xenografts

Unknown Cardionogen-1, cardionogen-2and cardionogen-3

Cardionogens can rescue cardiac cell deficiency caused by WNT8 in zebrafish

Activator

GSK3β Indirubins (INO), SB-216763 and6-bromoindirubin-3′-oxime (BIO)

GSK3β inhibitors can either enhance or inhibit tumour growth and metastasis in acontext-dependent manner

AXIN2–CTNNB1association

SKL2001 Not reported

SFRP1 WAY-316606 Not reported

CTNNB1 Deoxycholic acid Enhances colorectal cancer cell proliferation and invasiveness

CTNNB1 stability Cpd1 and Cpd2 Not reported

Synergist

Unknown Oxepane-derived compounds Not reported

Metabotropic glutamatereceptors

Riluzole Decreases melanoma cell proliferation in combination with WNT3A, and decreasesmelanoma metastasis in a xenograft model

*A full table with references can be found in Supplementary information S2 (table). APC, adenomatous polyposis coli; CBP, CREB-binding protein; CK1α, caseinkinase 1α; CTNNB1, β-catenin; DVL, Dishevelled; FAP, familial adenomatous polyposis; FZD, Frizzled; GSK3β, glycogen synthase kinase 3β; LGR5, leucine-richrepeat containing G protein-coupled receptor 5; SFRP1, secreted frizzled-related protein 1.

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cancer progression in a context-dependent manner. Takentogether, these studies argue that aberrations in WNT sig-nalling cannot be targeted using a single universal strat-egy, but rather that clinical decision-making should beinformed by our increasingly detailed understanding ofthe context-dependent roles of WNT signalling in cancer.Future studies aimed at determining the mechanisms thatcontrol this context-dependency will be necessary to iden-tify signalling nodes that could be targeted by therapeu-tic interventions. Given the interconnectedness of WNTsignalling with other oncogene and tumour suppressorpathways, it will also be important for future research tofocus on further unravelling the mechanisms of crosstalkbetween WNT pathways and related signalling networks.

In the past decade, we have witnessed an explosionin the development of strategies for targeting WNT sig-nalling. Many synthetic modulators of WNT signalling— including small molecules, peptides and blocking

antibodies — show great promise in animal modelsof several different cancers. Importantly, activating orinhibiting WNT signalling pathways alone is unlikelyto result in a substantial improvement in disease pro-gression owing to the co-activation of numerous onco-genic pathways in most cancers. Further research isclearly necessary to not only optimize these reagentsfor applications in animals and eventually in humanpatients, but also to further explore the potential valueof combinatorial therapies. Studies aimed at identify-ing the genetic factors and biomarkers that can predictresponses to treatment with WNT pathway modula-tors, either alone or in combination with other thera-pies, will be an important next step in determining theutility of these potential new therapies. Despite thesefuture challenges, these pioneering studies suggest thattargeting WNT signalling pathways in cancer patientswill be possible in the near future.

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AcknowledgementsWe thank B. Major for his helpful comments on this manu-script. We apologize to our colleagues whose work could not

be cited here due to space limitations. R.T.M. is an investiga-tor of the Howard Hughes Medical Institute.

Competing interests statementThe authors declare no competing financial interests.

FURTHER INFORMATIONRandall T. Moon’s homepage:

http://faculty.washington.edu/rtmoon

Catalogue of Somatic Mutations in Cancer (COSMIC)

database: http://www.sanger.ac.uk/genetics/CGP/cosmic

SUPPLEMENTARY INFORMATIONSee online article: S1 (table) | S2 (table)

ALL LINKS ARE ACTIVE IN THE ONLINE PDF

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