Targeting Wnt signaling in Colon Cancer Stem Cells · 18/12/2010 · observations highlight the...

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research

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Molecular Pathways

Targeting Wnt signaling in Colon Cancer Stem Cells

Felipe de Sousa E Melo1, Louis Vermeulen1, Dick Richel2 and Jan Paul Medema1,*

1Laboratory for Experimental Oncology and Radiobiology (LEXOR), Center for Experimental

Molecular Medicine (CEMM), Academic Medical Center (AMC), Amsterdam, the Netherlands. 2Department of Medical Oncology, Academic Medical Center (AMC), Amsterdam, the Netherlands

*to whom correspondence should be addressed ([email protected])

Research. on March 20, 2021. © 2010 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Author Manuscript Published OnlineFirst on December 15, 2010; DOI: 10.1158/1078-0432.CCR-10-1204

http://clincancerres.aacrjournals.org/


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Abstract

The identification of cancer stem cell (CSC) populations in virtually all tumour types

has widespread clinical consequences. CSCs are suggested to be the only cells within

malignancies endowed with tumourigenic capacity and are therefore directly

implicated in therapy resistance and minimal residual disease. The genetic and

molecular mechanisms sustaining CSCs are only currently emerging. For instance,

aberrant activation of the Wnt signaling pathway is crucial for many cancer types and

especially those of the gastrointestinal tract. Indeed, Wnt signaling activity was shown

to designate colon CSCs and is therefore an attractive target for new therapeutics.

Here we review some of the latest developments that have been achieved to inhibit

the Wnt pathway in the context of colon CSCs. Moreover, we discuss some of the

pitfalls that can be anticipated and present new opportunities for therapeutic

intervention.





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Background

Colorectal cancer is the second cause of cancer related death in the Western world

with an incidence of more than 1 million new cases each year (1). Colorectal cancer is

one of the best-studied malignancies and despite recent advances in chemotherapies

that have improved survival, patients with late stage disease still have poor prognosis

and the overall mortality of the disease is around 40% (2). Much of our understanding

on the histo-pathological and molecular processes underlying the transition from a

normal epithelium to an invasive adenocarcinoma relies on seminal work from Fearon

and Vogelstein (3). They have described the so called “adenoma-carcinoma

sequence” as the stepwise accumulation of genetic and epigenetic changes in

oncogenes and tumour suppressor genes.

More recent insights from the cancer stem cell (CSC) field have re-shaped our view

of malignancies. The colorectal cancer stem cell model poses an interesting

framework in which tumours are hierarchically organized tissues with CSCs at the top

of the hierarchy driving tumour growth and progression (4). CSC are defined as the

cells that are endowed with both self-renewal and multi-lineage differentiation

potential (4-5) and as such are believed to clonally expand and repopulate the various

types of differentiation lineages present within the tumour. The more differentiated

progeny have lost their self-renewing capacity and are thought to be dispensable for

tumour maintenance. The CSC theory therefore has dramatic consequences for the

way we perceive cancer initiation and progression and currently serves as a basis for

targeted therapies. However, the development and clinical use of effective therapies

will depend on accurate understanding of the molecular processes regulating CSCs.

Here we review the latest insights on molecular pathways regulating colon CSCs with

specific emphasis on the Wnt signaling cascade. Then we will discuss the rationale

behind targeting Wnt signaling and the potential caveats to this approach.

The Wnt canonical signaling pathway

The Wnt signaling cascade is conserved throughout the animal kingdom and,

depending on the context, plays various roles that encompass stem cell maintenance,

cell proliferation, differentiation and apoptosis (reviewed in ref. 6). The canonical

pathway is mainly regulated at the level of β-catenin, a protein kept under low

cytoplasmic concentration by the destruction complex. The latter contains the tumour





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suppressor protein Adenomatous Polyposis Coli (APC), two kinases: Casein kinase 1

(CK1) and glycogen synthase kinase 3β (GSK3-β), and Axin2 that scaffolds the

complex together. In the absence of Wnt ligands, the membrane receptor complex

formed by Frizzled (Fzd) and LRP5/6, are not engaged and CK1 and GSK3β

phosporylate β-catenin at specific serine and threonine residues, priming its

recognition by the U3 ubiquitin ligase β-TrCP. As a consequence, β-catenin is

ubiquitinated and targeted for proteosomal degradation (Fig. 1.A and ref. 7).

Upon binding of Wnt ligands to the receptors, the destruction complex is dissolved by

an ill-defined mechanism (8) and β-catenin is no longer degraded, which leads to its

accumulation in the cytosol and subsequently translocation into the nucleus. There it

associates with the LEF/TCF (lymphoid enhancer factor/T cell factor) family of

transcription factors converting them from repressors to activators of transcription.

These nuclear events require in a first step displacement of the co-repressor Groucho

(9) and subsequently recruitment of the histone acetylase CBP/p300 and co-activators,

like Pygopus (PYG) and BCL9 (10). This triggers a complex transcriptional program

that directs cell fate, cell proliferation and stem cell maintenance (Fig. 1.B). Important

Wnt target genes include c-MYC (11), Axin2 (12) and ASCL2 (13) that serve

important functions in various stages during embryogenesis, but also during organ

homeostasis and colorectal cancer development.

Wnt signaling in homeostasis of the gut

The role of Wnt signaling in adult tissue homeostasis is best illustrated in the gut

where a gradient of Wnt signaling activity is required for the organization and

patterning of the intestinal tract (reviewed in ref. 14). Wnt signaling components are

present throughout the crypt villus-axis (15), active canonical signals are critical to

maintain the stem cell compartment, located at the bottom of the crypt. Blockade of

Wnt signaling, either by artificial deletion of TCF4 or over-expression of the Wnt

antagonist Dickkopf-1 (DKK1), results in loss of epithelial cell proliferation and

intestinal tissue structure (16-17). Furthermore, positioning of stem and differentiated

cells throughout the crypt-villus axis is orchestrated by the EphB2 and B3 receptors,

which are also TCF4 targets (18). Using the TCF4-induced transcriptional program

combined with specific localization of identified Wnt target genes to the bottom of the

crypt, Barker et al. identified LGR5 as stem cell marker for both intestine and colon

(19). Other Wnt targets exemplify the functional role of Wnt signaling in stem cell





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maintenance. For instance, ectopic expression or, reciprocally, conditional deletion of

ASCL2, a transcription factor that is also restricted to the crypt results in intestinal

hyperplasia and loss of the stem cell compartment respectively (20). Although beyond

the scope of this review, it is important to note that other morphogenetic pathways,

such as BMP and Notch signaling, are, in conjunction with Wnt signaling, important

regulators of gut homeostasis.

Deregulation of the Wnt pathway and intestinal tumours

Given its fundamental role in homeostasis in adult tissue, it is not surprising that

deregulation of the Wnt pathway is associated with various pathological states

including various types of cancer (21-22). Indeed, loss of function of Wnt

components is critically involved in the pathogenesis of colorectal cancer (23).

Inactivation of the APC gene or activating mutations of β-catenin is reported in

virtually all patients presenting with colorectal cancer (24) and is believed to be the

critical initiating step in malignant transformation (25). Although of various nature,

those mutations ultimately result in stabilization of β-catenin and perpetual activation

of the Wnt transcriptional program even in the absence of any extracellular signals

(Fig. 1.C).

Interestingly, although most patients contain constitutively activating

mutations of the Wnt pathway, such tumours often still reveal a certain degree of

regulation of the pathway. There are several lines of evidence to support this. First,

the histopathological observation that not all tumour cells deficient for APC display

homogeneous nuclear β-catenin staining, a surrogate for Wnt signaling activity (26-

27). This observation has been dubbed the “β-catenin paradox”. Second, the two-hit

hypothesis that normally results in inactivation of a tumour suppressor gene is thought

to be independent events. However, for APC this does not seem to be the case since it

has been shown that the type of germline APC mutation that is present in familial

adenomatous polyposis (FAP) patients influences the nature of the second, ‘somatic’

hit in the APC gene (28). Importantly, this never results in a complete loss of function

of the protein and suggests a fine tuned balance of Wnt activity that is required for

optimal cell transformation (29). This principle is often quoted as the “just-right”

signaling model. Finally, recent observations from our laboratory have demonstrated

that Wnt signaling activity in colon cancer is also characterized by a gradient where

colon CSCs are functionally marked by a highly active Wnt signaling pathway,





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whereas the differentiated progeny of these cells show markedly lower levels of

activity. This gradient is at least in part orchestrated by the microenvironment. These

observations highlight the role of Wnt signaling pathway regulation in colorectal

cancer and its role in colon-CSC features. As mentioned above, the CSCs theory has

widespread consequences on the rationale treatment of cancer. The relevance of Wnt

activity levels in defining these cells in colorectal cancer provides a potential new

interesting target. In this respect it has become increasingly clear that various types of

malignancies, aside from colorectal cancer, are dependent on sustained Wnt activity.

Therefore the therapeutic benefit of drugs successfully targeting Wnt signaling is

evident also for various cancer types. In this second part we review the current drugs

that are emerging especially for the treatment of colorectal cancer.

Clinical-Translational Advances

Targeting Wnt pathway components

An incredible collection of natural and synthetic compounds form the basis of intense

efforts in high-throughput drug screening programs. The past decades have seen

major advances in understanding the molecular framework of Wnt signaling that

provide an optimal platform for testing these libraries of compounds (30). In 2009,

Lum et al. screened diverse chemical libraries and identified two classes of molecules

with Wnt inhibitory features. The first class acts primarily at the level of Wnt ligands

production by specifically targeting Porcupine (PORCN), an acyltransferase that adds

a palmitoyl group to Wnt proteins, an essential step for their secretion. The second

class regulates Axin2 stability and importantly also targets β-catenin degradation in

the presence of APC mutations (31). Additionally, another recent study has

highlighted the role of the poly-ADP-rybosylating enzymes Tankyrase 1 and 2

(TNKS) in promoting Axin2 degradation. Enzymatic inhibition of TNKS by XAV-

939 is able to stabilize Axin2 and promotes degradation of β-catenin (32). Although

of potential interest for various Wnt signaling dependent malignancies, the benefit for

colorectal cancer is questionable as the first class of inhibitors will in theory be

inefficient when APC mutations render the tumour Wnt ligand independent (33).

However, as mentioned, APC mutations rarely represent complete null mutations. In

agreement, Wnt ligands are expressed in various CRC cell lines and blockade of

Wnt1 with monoclonal antibodies can trigger apoptosis in cell lines bearing APC as





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well as β-catenin mutations (34-35). Conversely, Wnt natural inhibitors such as

secreted Frizzled related proteins (SFRPs) are often methylated and silenced in

primary tumours (36). These proteins share similarities with Wnt cell surface Fzd

receptors, and can prevent their binding with Wnt ligands and subsequent activation

of the pathway (37). Similar to inhibition of Wnt1, re-expression of SFRP in CRC cell

lines or their epigenetic re-activation results in decreased Wnt activity as well as cell

death (36). These insights clearly support a rational for targeting the extracellular

machinery upstream of the destruction complex. Therefore, an antibody targeting

approach against Wnt ligands and/or blockade of the Frz receptor signaling might

provide an interesting therapeutic avenue to explore (38). From a more fundamental

biological perspective, it also supports the notion that full activation of Wnt signaling

cannot be explained by APC mutations alone, or alternatively, that Wnt ligands

activate crucial non-canonical Wnt signaling routes.

The transcriptional program that initiates malignant transformation requires nuclear

localization of TCF/β-catenin where abrogation of this complex can block the target

gene expression and cell growth in vitro (17). Therefore targeting the TCF/β-catenin

nuclear complex holds as well great promises for successful therapy. The recruitment

of transcriptional co-activators such as PYG, BCL9 and CBP/p300 are well

documented and their induced absence is expected to impinge a proper Wnt

activation. As a proof of principle, Kahn et al. screened for TCF/β-catenin inhibitors

and found the leading compound ICG-001 that specifically targets and inhibits the co-

activator CBP (39). Treatment of CRC cell lines bearing APC or β-catenin mutations

with this compound induces dose dependent cell death, while normal colonic

epithelial cells are resistant. The effect is also seen in the APCmin mouse model and

in tumour xenografts. As a result of this ICG-001 is expected to shortly enter in

clinical phase I trials.

Indirect targeting of the Wnt signaling cascade

Although of great potential, most Wnt inhibitors are still in preclinical testing or

developmental stage. Additionally, given the fact that Wnt signaling is such an

important pathway involved in regulation of tissue homeostasis, interference with

crucial components of this cascade is predicted to be associated with serious adverse

events. For example, imbalance of intestinal and hematopoietic homeostasis is a

predictable bystander effect of non-specific Wnt inhibition (40). This requires





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anticipation in designing drugs that that will offer great specificity and a certain

therapeutic window between normal stem cells and CSCs. For example, drugs that

have been studied in other clinical settings also have substantial therapeutic impact

partially dependent on their Wnt inhibitory properties. The use of Nonsteroidal anti-

inflammatory drugs (NSAIDs), like sulindac and aspirin, has been suggested in a

number of epidemiological studies to have a chemoprotective role in colorectal cancer

(41). Preclinical studies have shown a correlation between efficacy of

chemoprevention and the Wnt modulatory effects of these compounds (42). NSAIDs

have complex mode of actions and only part of them converged to an inhibition of the

cyclooxygenase (COX) enzymes (43). COX-2 expression is seen increasingly in early

stages of colorectal cancer (44). This enhanced expression drives the production of

the prostaglandine E2 (PGE2), which mediate tumour progression, angiogenesis and

metastasis (45). Mechanistically, COX-2 induced PGE2 can prevent β-catenin

degradation by inhibiting both GSK-3β and Axin2 and as a result activate Wnt

signaling (Fig. 2.B and refs. 46-47). The inhibition of COX-2 can only partially

account for the beneficial effect of NSAIDs and COX-2 specific inhibitors (Coxibs),

such as celocoxib and rofecoxib on colorectal cancer. NSAIDs and celocoxib can also

induces colorectal cancer cell death independently of COX-2 expression (48). In

agreement, NSAIDs deprived of COX-2 inhibitory capacities also have an effect on

colorectal cancer (49). For example, growth inhibition via up-regulation of the cell

cycle inhibitors p21Waf1 is one of COX-2 independent mode of actions of celocoxib

(50). Another interesting mechanism involves the tyrosine kinase receptor C-MET

(54). C-MET also known as hepatocyte growth factor (HGF) receptor is known to

influence Wnt signaling. Binding of HGF to its receptor induces dissociation of

membrane bound β-catenin from the E-cadherin complexes (51). Additionally, C-

MET activation can activate PI3kinase signaling and subsequent phosphorylation and

inactivation of GSK-3β (27, 52). As a result, β-catenin that is part of the destruction

complex is no longer degraded but stabilized. Moreover, β-catenin phosphorylation

on ser552 by pAKT/PKB is a nuclear translocation mark (Fig. 2.A and ref. 53) also

triggered by PI3kinase activation. These concomitants events initiated by HGF

ultimately boost β-catenin levels in the cytosol and nucleus and therefore regulate

Wnt activity. On the other hand, celocoxib can block C-MET dependent

phosphorylation of various substrates that are accompanied by an increase in GSK-3β





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activity. Thus resulting in β-catenin degradation and in Wnt signaling inhibition (54).

Despite other modes of action of coxibs that requires further clarification, celocoxib is

approved by the food and drug administration (FDA) for the treatment of familial

adenomatous polyposis (FAP) (55). It is however important to note that the potential

benefit of coxibs in colorectal cancer prevention in the general population is

hampered by cardiovascular side effects (55-56).

As described, small molecule inhibitors and natural compounds have been identified

to have potential therapeutic value against cancers associated with aberrant Wnt

signaling either by direct or indirect mechanisms. However, their lack of specificity,

our lack of knowledge of their precise targets and working mechanism or their

adverse side effects has precluded the start of clinical trials. Identification of these

targets molecules and determination of the precise mechanism of action of these

agents may provide novel targets.

Future perspectives and concluding remarks

The discovery and generation of new cancer drug regimens requires thorough

understanding of the basic biological events that drive cancer initiation, progression

and maintenance. As the CSCs theory explains part of these processes, an important

effort has been made to scrutinize and define their regulation that will yield an

invaluable new source of therapeutic strategies. It is however important to integrate

the regulation of the CSCs in a more general context. As in normal adult tissue where

stem cell resides in specific protective microenvironment or niche, tumours are also

influenced by micro-environmental cues and are increasingly perceived as aberrant

but highly organized tissues (57-58).

Indeed, we and others have shown that such niche requirements are also found in

malignancies where they contribute to the CSC phenotype (27, 59). More importantly,

when micro-environmental stimuli, such as HGF that is predominantly secreted by the

tumour stroma, are applied on the more differentiated tumour cells, these cells

undergo a de-differentiation program and revert back to CSCs (27). This plasticity of

differentiated cancer cells suggests a more dynamic interpretation of the CSC model

that has crucial implications, especially for therapies that are aimed at specifically

targeting the CSC fraction, which would be counteracted by re-population of the CSC

pool. On a more positive note, this interaction would provide a complete novel





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therapeutic possibility. In light with this, small molecule inhibitors and/or monoclonal

antibody developed to target the C-MET receptor, such as PF-02341066 might prove

efficacy for cancer treatment and are in clinical trials (60-61). In the near future, these

new playgrounds of drug development will aim to tackle the various CSCs regulatory

axes and will hopefully yield efficient therapy regimens resulting in improved clinical

outcome.

Acknowledgements

The authors would like to thank Tijana Borovski for critical comments on the

manuscript. This work was supported by a VICI grant from the Netherlands

Organisation for Scientific Research and a Dutch Cancer Society (KWF

Kankerbestrijding) grant (2009-4416) (to J.P.M.), an Academisch Medisch Centrum

(AMC) fellowship (to L.V. and F.d.S.M.).





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Figure. 1. The Wnt canonical pathway

A. In the absence of Wnt ligands, β-catenin is kept under low cytosolic level by the

destruction complex. This latter contains Axin2 and APC that present β-catenin to the

two kinases CK1 and GSK3-β, facilitating its phosphorylation at specific serine and

threonine residues. This primes β-catenin recognition by β-TRCP, which targets it for

proteosomal degradation. In the nucleus, TCF transcription factors are bound to co-

repressor (Groucho) and gene transcription is actively repressed (7). B. Wnt ligands

bound to Fzd and LRP5/6 co-receptors triggers formation of Dvl-Fzd complex and

phosphorylation of LRP by GSK3-β. This recruits the scaffolding protein Axin2 to

the co-receptors and as a result the destruction complex is dissolved (8). β-catenin is

therefore stabilized, can accumulate in the cytosol and subsequently translocate in the

nucleus where it converts TCF into a transcriptional activator. This step is mediated

by the displacement of the Groucho protein and recruitment of co-activators that

includes CBP, BCL9 and Pygopus (PYG) (10). This ensures efficient transcription of

genes that are important regulators of stem cell fate (LGR5, ASCL2), cell proliferation

(C-MYC), and as well negative regulators of the pathway (Axin2). C. Truncating

mutations in APC are frequently observed in colorectal cancer. As a result, the

destruction complex cannot properly form, which results in inefficient targeting of β-

catenin for degradation degradation. Therefore, β-catenin can accumulate and form

active transcription factor complexes with TCF proteins in the nucleus, even in the

absence of external signal. (Fzd, Frizzled; LRP, low-density lipoprotein receptor-

related protein; Dvl, Dishevelled; APC, Adenomatous polyposis coli; GSK3-β,

Glycogen synthase kinase 3 β; CK1, Casein kinase 1; TCF, T cell factor; CBP,

CREB-binding protein, β-TRCP, β-transducin repeat-containing protein).





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Figure. 2. Targeting Wnt signaling

A. HGF is mainly produced by stromal myofibroblasts. Binding to its receptor C-

MET triggers activation of PI3 kinase signaling and in turn AKT/PKB

phosphorylation. Activated AKT/PKB phosphorylates GSK3-β at a specific serine

residue which renders it inactive and unable to prime β-catenin for degradation (27-

52). Additionally, AKT/PKB phosporylates β-catenin at a specific serine residue

which enhances its nuclear translocation (53). Altogether, this contributes to an

increase in nuclear TCF-β-catenin complexes. B. Elevated levels of COX-2 are

observed in cancer cells (44). This results in increased prostaglandin PGE2

production (45). Via its receptor, PGE2 can efficiently prevent β-catenin degradation

by interfering with both GSK3-β and Axin2 function (46-47). A panel of, direct or

indirect, Wnt inhibitors (orange) and their molecular targets are also depicted. For

instance, IWR (31) stabilizes Axin2. Celocoxib inhibits COX-2 downstream signaling

(43) but also target the receptor C-MET (54). Tankyrase (TNKS) is a poly-ADP-

rybosylating enzyme that promotes Axin2 degradation and is targeted by XAV-939.

Monoclonal antibodies (R13 and R28) can block HGF/C-MET interaction (60).





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Published OnlineFirst December 15, 2010.Clin Cancer Res Felipe De Sousa E Melo, Louis Vermeulen, Dirk J Richel, et al. Targeting Wnt Signaling in Colon Cancer Stem Cells

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