ReVIeWS

Mutations and mechanisms of WNT

pathway tumour suppressors in cancer
Jeroen M. Bugter , Nicola Fenderico and Madelon M. Maurice ✉

Abstract | Mutation-​induced activation of WNT–β-​catenin signalling is a frequent driver event in

human cancer. Sustained WNT–β-​catenin pathway activation endows cancer cells with sustained
self-​renewing growth properties and is associated with therapy resistance. In healthy adult stem
cells, WNT pathway activity is carefully controlled by core pathway tumour suppressors as well
as negative feedback regulators. Gene inactivation experiments in mouse models unequivocally
demonstrated the relevance of WNT tumour suppressor loss-​of-​function mutations for cancer
growth. However, in human cancer, a far more complex picture has emerged in which missense or
truncating mutations mediate stable expression of mutant proteins, with distinct functional and
phenotypic ramifications. Herein, we review recent advances and challenges in our understanding
of how different mutational subsets of WNT tumour suppressor genes link to distinct cancer types,
clinical outcomes and treatment strategies.

Stem cell
An undifferentiated cell that
divides indefinitely to renew
itself and produce progenitors
that differentiate into
specialized cell types to
replenish lost cells in the
tissue during homeostasis
and repair.
Missense mutations
Single base pair substitutions
in the genome that result in a
change in the codon, leading to
the encoded protein bearing a
single amino acid substitution.
Nonsense mutations
Single base pair substitutions
in the genome that result in an
early stop codon, leading to a
prematurely truncated protein
variant or nonsense-​mediated
mRNA decay.
Oncode Institute and
Department of Cell Biology,
Center for Molecular Medicine,
University Medical Center
Utrecht, Utrecht, Netherlands.
✉e-​mail: m.m.maurice@
umcutrecht.nl
https://doi.org/10.1038/
s41568-020-00307-​z
Nearly 40 years ago, the first WNT gene was discovered1.
This discovery unveiled WNT1 as the gene responsible
for driving mammary tumorigenesis in mice upon inser-
tion of the mouse mammary tumour virus (MMTV)1.
In the following two decades, most of the key components
of the WNT pathway were unravelled. Concurrently,
these discoveries signified prominent roles for WNT sig-
nalling in the control of embryogenesis as well as cancer
development2. Later work uncovered WNT signalling as
a primary driving force for adult stem cell self-​renewal
and cell-​fate specification in the intestine, liver, skin and
many other organs 3–7. During tissue homeostasis,
WNT pathway activity is tightly controlled by various
tumour suppressors that function either as core compo­
nents or act in negative feedback loops8–14. Mutation or
silencing of WNT tumour suppressor genes has been
identified as a frequent occurrence in a wide variety of
cancer types15. In line with these observations, persistent
WNT–β-​catenin pathway activation was found to endow
cancer cells with self-​renewing growth properties and,
furthermore, is linked to therapy resistance16,17.
Large-​scale sequencing efforts have made it clear
that genetic alterations in cancer are highly diverse
and complex. Whereas mutations in oncogenes often
display mutational hot spots that bring about protein
overactivity, the landscape of mutations in tumour
suppressor genes usually comprises a mixture of dele-
tions and missense mutations, nonsense mutations and
frameshift mutations that are found scattered over the
whole length of the gene, complicating functional inter-
pretation. Thus, these observations have posed a chal-
lenge to unambiguously distinguish passenger mutations
from driver mutations and predict whether and how these
mutations generate vulnerabilities that can be exploited
by cancer treatment18. Furthermore, mutations that hit
a single pathway may display noticeable tissue or cancer
subtype specificity, or show cooperative effects with
mutations that deregulate alternative signalling pathways
in the same tumour, further enhancing complexity and
impeding a mechanistic understanding of how genetic
alterations contribute to cancer growth.
This Review focuses on recent advances in our under-
standing of how various classes of mutations in WNT
pathway tumour suppressors contribute to cancer deve­
lopment. An overview is provided of how different WNT
pathway mutations link to tissue specificity and clini-
cal progression of disease. We discuss consequences of
loss-​of-​function (LOF) mutations in preclinical models and
human patients and describe how the investigation of
other classes of patient-​derived mutations have unveiled
novel mechanisms by which WNT pathway tumour sup-
pressor mutations drive cancer growth and progression.
Finally, we highlight the need for a more comprehensive
understanding of the common principles by which WNT
pathway mutagenic events drive carcinogenesis to enable
improved applications of precision cancer medicine and
the design of more effective therapies.
WNT–β-​catenin signalling
WNT-​mediated receptor activation can direct signalling
along distinct pathways that are roughly divided into
β-​catenin-​dependent and β-​catenin-​independent signal-
ling routes. Herein, we focus on the β-​catenin-​dependent
pathway that is prominently linked to the development

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a Off On
LRP5
FZD WNT
or LRP6

DVL DVL
CK1 GSK3β CK1 APC AXIN1
GSK3β
GSK3β β-TrCP
AXIN1 AXIN1 CK1
β-Catenin β-Catenin Ub
APC APC
P P P P P P Ub β-Catenin
β-TrCP β-Catenin
Ub
Destruction
complex

β-Catenin • AXIN2
• RNF43
TCF TCF • ZNRF3
Nucleus • LGR5

b Negative feedback WNT potentiation

RSPO
RNF43 WNT RSPO WNT
or ZNRF3

1 Ub DVL
Ub LGR APC AXIN1
AXIN2 Ub
2 Endosome GSK3β
CK1
CK1 GSK3β
AXIN1/2 β-Catenin
β-Catenin
APC
P P P β-Catenin
β-Catenin
TCF β-Catenin TCF
Lysosomal Lysosomal
degradation degradation
Frameshift mutations
Mutations by which one or
c WNT-independent WNT-hypersensitive
multiple base pairs are inserted
in or deleted from the genome WNT
resulting in a change of the
reading frame of the gene,
leading to a truncated protein
variant with an incorrect amino DVL
acid sequence downstream
CK1 GSK3β APC AXIN1
of the frameshift or nonsense- DVL β-TrCP GSK3β
mediated mRNA decay. AXIN1/2 CK1
β-Catenin
Passenger mutations APC
P P P β-Catenin
Mutations that do not endow
β-TrCP
a competitive advantage but β-Catenin
coincide with a driver mutation. β-Catenin β-Catenin β-Catenin
Typical cancer genomes β-Catenin β-Catenin
comprise 102–106 passenger β-Catenin β-Catenin
mutations.
TCF TCF
β-Catenin β-Catenin
Driver mutations
Mutations that endow tumour
cells with a growth or survival
advantage over cells without of cancer16,19. A central event in this signalling cascade is it is ubiquitylated by the E3 ligase SKP1-​CUL1-​F-​box
this mutation. On average,
the regulated proteolytic turnover of the transcriptional protein (SCF)–β-​transducin repeat-​containing protein
cancer genomes comprise
four or five driver mutations. co-activator β-​catenin. In resting cells, the pool of cyto- (β-​TrCP) and dispatched to the proteasome to complete
solic β-​catenin is kept low by the activity of a multisub- its degradation10,20–27 (Fig. 1a).
Loss-​of-​function (LOF) unit destruction complex, composed of the tumour WNT-​mediated stimulation of cells is initiated by
mutations suppressors AXIN1 and adenomatosis poly­posis coli the formation of a trimeric complex between WNT and
Genomic mutations resulting
in an (partially) inactive gene
(APC) and the kinases casein kinase 1 (CK1) and glyco­ its receptors, Frizzled (FZD) and low-​density lipopro-
product. They often involve gen synthase kinase 3β (GSK3β). This protein complex tein receptor-related protein 5 (LRP5) or LRP6, at the
loss of heterozygosity. captures and phosphorylates β-​catenin, after which cell surface. Receptor activation mediates recruitment

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◀ Fig. 1 | WNT signalling pathway regulation and mutation routes to WNT-independent
or WNT-hypersensitive growth states in cancer. a | Left: in the absence of WNT
ligands, cytosolic β-​catenin is continuously degraded by a destruction complex
comprising the oligomerizing scaffold proteins adenomatous polyposis coli (APC)
and AXIN1 and the associated kinases glycogen synthase kinase 3β (GSK3β) and casein
kinase 1 (CK1). The kinases phosphorylate β-​catenin, marking it for ubiquitylation by
the E3 ligase SKP1-​CUL1-​F-​box protein (SCF)–β-​transducin repeat-​containing protein
(β-​TrCP) and subsequent proteasomal degradation. Right: WNT proteins bind and
activate their cognate receptor Frizzled (FZD) and co-​receptor low-​density lipoprotein
receptor-related protein 5 (LRP5) or LRP6. Next, the cytosolic protein Dishevelled (DVL)
is recruited to the plasma membrane by the activated receptors, where it promotes
multimerization of the receptor complex and recruits components of the destruction
complex, ultimately leading to inhibition of β-​catenin proteolysis. As a consequence,
cytosolic β-​catenin accumulates and translocates to the nucleus where it promotes
the transcription of WNT target genes in association with T cell factor/lymphoid
enhancer-​binding factor (TCF/LEF) transcription factors. b | Left: WNT target gene
negative feedback regulators ring finger protein 43 (RNF43) and zinc and ring finger
protein 3 (ZNRF3) ubiquitylate FZD proteins and mark these receptors for endocytosis
and lysosomal degradation (step 1). In addition, RNF43 facilitates the restoration of
destruction complex activity (step 2). WNT target gene negative feedback regulator
AXIN2 helps the formation of new destruction complexes. Whether and how restoration
of destruction complex activity involves relocation from the receptor complex to the
cytosol remains unknown (dashed arrow). These events lead to attenuation of WNT
signalling. Right: in stem cells, R-​spondins (RSPOs) bind to their cognate receptors
leucine-​rich repeat-​containing G protein-​coupled receptor 4 (LGR4), LGR5 or LGR6
and the extracellular domain of RNF43 or ZNRF3. This complex facilitates RNF43 and
ZNRF3 clearance from the plasma membrane, thereby potentiating WNT signalling.
c | Molecular mechanisms of aberrant WNT signalling pathway activation mediated
by cancer mutations. Tumour suppressor mutations (left) abrogate destruction complex
activity to drive WNT-​independent growth or (right) abrogate negative feedback at the
receptor level to drive WNT-​hypersensitive growth. P, phosphorylation; Ub, ubiquitin.
of the effector protein Dishevelled (DVL), which in turn
recruits AXIN1 and the other core destruction complex
components to the plasma membrane28–35. Together,
these events abrogate destruction complex-​mediated
degradation of β-​catenin, allowing it to accumulate
and translocate to the nucleus to bind the T cell factor/
lymphoid enhancer-​binding factor (TCF/LEF) family of
transcription factors and drive the transcription of WNT
target genes36,37 (Fig. 1a).
To prevent excessive signalling, WNT-induced
res­ponses are balanced by numerous negative feedback
regulators. The WNT target gene AXIN2 (also known as
conductin), the RNA product of which is commonly used
as a marker for WNT pathway activity, helps to increase
cytosolic destruction complex activity and downregulate
β-​catenin-​mediated transcription11,12,38. Furthermore,
induced expression of the membrane-​bound E3 ligases
ring finger protein 43 (RNF43) and zinc and ring finger
protein 3 (ZNRF3) mediates ubiquitylation of WNT
receptors, which drives their internalization and lyso-
somal degradation, thereby attenuating the sensitivity
of cells to incoming WNTs13,14. Additionally, RNF43
was found recently to perform a secondary suppressor
role, preventing β-​catenin-​mediated transcription by
promoting destruction complex activity, through an
incompletely understood mechanism that involves
regu­latory interactions of the RNF43 cytosolic tail with
CK1 (ref.39) (Fig. 1b). However, within the adult stem cell
niche, negative feedback by RNF43 and ZNRF3 is coun-
teracted locally by secreted proteins of the R-​spondin
(RSPO) family13. By forming a complex with members
of the leucine-​rich repeat-​containing G protein-​coupled
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receptor (LGR) family and RNF43 or ZNRF3, RSPO
promotes the clearance of RNF43 and ZNRF3 from
the cell surface13,40,41 (Fig. 1b). Additionally, a subset of
RSPO family members promotes clearance of RNF43
and ZNRF3 via an LGR-​independent mechanism that
involves binding to heparan sulfate proteoglycans
(HSPGs)42–45. Thus, RSPO activity allows WNT recep-
tors to accumulate at the cell surface and induce high
levels of β-​catenin-​mediated transcription required for
stem cell maintenance.
WNT tumour suppressor mutations
Driver routes for β-​catenin activation
The primary output of driver mutations in WNT pathway
tumour suppressors is unwarranted β-​catenin-​mediated
transcription, which activates a gene programme that
promotes stemness, proliferation and survival 16,19.
Excessive β-​catenin activity can be achieved by two
major mutational driver routes. Mutational inactivation
of the β-​catenin destruction complex is the archetypal
mode of WNT pathway activation in cancer, exemplified
by inactivating mutations in APC, AXIN1 and AXIN2 or
activating mutations in β-​catenin (encoded by CTNNB1)
itself  8,46–48. These events are generally assumed to drive
a state of WNT-​independent tumour growth (Fig. 1c).
More recently, misregulation of WNT receptor abun-
dance emerged as a second route to carcinogenesis.
This oncogenic route is initiated by loss of RNF43 and
ZNRF3, thus eradicating the negative feedback loop that
normally drives WNT receptor endocytosis in adult
stem cells14. Of note, RNF43- and ZNRF3-​depleted cells
have lost their requirement for RSPO and thus display
aspects of niche-​independent growth, a hallmark of
cancer cells14,49. An alternative pathway by which cancer
cells may avoid the activities of RNF43 and ZNRF3 is
by acquired RSPO overexpression50,51. As a consequence,
these cancers display an overabundance of cell surface
WNT receptors, which mediates ligand hypersensi­
tivity and promotes WNT-​dependent tumour growth13,14
(Fig. 1c).
Tissue specificity
Although genetic alterations in the WNT pathway occur
in most cancer types, mutations in individual WNT sig-
nalling components show a substantial level of tissue
specificity. Examination of the frequencies of mutations
in individual WNT pathway components across 33 spo-
radic cancer types listed in The Cancer Genome Atlas
(TCGA) reveals that endometrial, skin and stomach
cancer collect mutations in various core components,
whereas other cancer types are more selective (Fig. 2a).
In colorectal cancer (CRC; n = 594), the largest fraction
of sporadic tumours accumulates APC mutations (67%),
followed by lower fractions of RNF43 (8%), CTNNB1
(6%) and AXIN2 (5%) mutations. By contrast, liver
cancer cases (n = 372) preferentially acquire CTNNB1
(25%) and AXIN1 (8%) mutations, whereas pancreatic
cancers (n = 184) favour mutations in RNF43 (6%) and
adrenocortical cancer (n = 92) links to mutations in
ZNRF3 (20%) or CTNNB1 (15%). Of note, the contri-
bution of WNT pathway alterations to cancer develop-
ment might be larger than reflected in TCGA database.
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a b APC RNF43 ZNRF3

APC Colorectal adenocarcinoma 66.55%

Uterine corpus endometrial carcinoma 14.34% CMS1
Skin cutaneous melanoma 14.25%
CMS2
Stomach adenocarcinoma 13.38%
Bladder urothelial carcinoma 7.04% CMS3
RNF43 Uterine corpus endometrial carcinoma 15.28% CMS4
Stomach adenocarcinoma 10.20%
Colorectal adenocarcinoma 7.90% c APC RNF43 ZNRF3
Pancreatic ductal adenocarcinoma 5.95%
ZNRF3 Adenocortical carcinoma 20.43%
Uterine corpus endometrial carcinoma 6.23%
Skin cutaneous melanoma 5.35%
AXIN1 Liver hepatocellular carcinoma 7.77%
Uterine corpus endometrial carcinoma 6.42%
Deep deletion
AXIN2 Uterine corpus endometrial carcinoma 7.36% AXIN1 AXIN2 CTNNB1
Colorectal adenocarcinoma 5.04% In-frame
mutation
CTNNB1 Liver hepatocellular carcinoma 25.47%
Missense
Uterine corpus endometrial carcinoma 25.47%
mutation
Adenocortical carcinoma 15.05%
Skin cutaneous melanoma 6.68% Truncating
Stomach adenocarcinoma 6.68% mutation
Colorectal adenocarcinoma 5.71% Gene fusion

Fig. 2 | Frequencies and types of WNT pathway mutation in human cancer types. Data source: Memorial Sloan
Kettering Cancer Center (MSKCC) cBioPortal196. a | Frequency of mutation or deep deletion of the indicated WNT pathway
genes per The Cancer Genome Atlas (TCGA) study. Frequencies >5% are shown. b | Consensus molecular subtypes (CMSs)
of TCGA colorectal cancer samples that have a mutation in adenomatous polyposis coli (APC), ring finger 43 (RNF43) or zinc
and ring finger 3 (ZNRF3). Samples without a CMS label were left out. CMS classification was derived from the Colorectal
Cancer Subtyping Consortium (CRCSC)66. c | Distribution of deep deletions, in-​frame mutations, missense mutations,
truncating mutations or gene fusions found in all TCGA Pan-​Cancer studies for APC, RNF43, ZNRF3, AXIN1, AXIN2 and
CTNNB1 (encoding β-​catenin).

Promoter hypermethylation
A common mechanism of
gene silencing, mediated by
methylation of CpG islands
in the promoter region. This
mechanism is frequently
misused by cancer cells to
turn off the expression of
(tumour suppressor) genes.
Familial adenomatous
polyposis
(FAP). An autosomal dominant
inherited condition that
involves extensive polyp
formation in the colon and
a high risk of colorectal
cancer, caused by a
heterozygous germline
mutation in adenomatous
polyposis coli (APC). A
second-​hit mutation or loss
of heterozygosity precedes
malignant polyposis.
Chromosomal instability
(CIN). Genomic instability
that involves frequent
mis-​segregation of
chromosomes during
cell division, resulting
in gain or loss of entire
chromosomes or sections
of them.
For instance, the number of identified RNF43 muta-
tions appears under-​represented owing to incomplete
calling of frameshift mutations that display similarity
to technical DNA slippage errors52. Furthermore, WNT
path­way regulators may be downregulated by promoter
hypermethylation, as shown for AXIN1 in a large frac-
tion of non-​small-​cell lung cancer (NSCLC) cases (43%,
29/67)53, as well as for ZNRF3 in BRAF-​mutant CRC
(72%, 36/50)54.
Tumour subtypes and prognosis
Although WNT tumour suppressor mutations all
converge on enhancing β-​catenin activity, emerging
evidence indicates that the functional consequences
of different WNT pathway mutations are not equal.
Large-​s cale cancer genome profiling revealed that
specific mutations in WNT pathway components may
occur either early or late during cancer development,
associate with selective cancer subtypes and differen-
tially correlate with clinical prognosis. Below, we discuss
recent insights obtained for two major cancer types.
Colorectal cancer. The prominent role of APC as a
gatekeeper gene was first revealed in 1991 by multiple
research groups who collectively showed that inactivat-
ing APC mutations cause familial adenomatous polyposis
(FAP), a syndrome characterized by extensive intesti-
nal polyp formation and a high risk of CRC develop-
ment46,55–57. These findings were substantiated by the
simultaneous identification of a large number of APC
mutations in sporadic CRCs58,59 (Fig. 2a). Molecular
characterization of tumour lesions at various stages of
development indicated that APC mutations are an early
and perhaps rate-​limiting event60. However, recent work
suggests that pre-​existing oncogenic KRAS mutations,
commonly found in the ageing human colon, might
mark preferred initiation sites for APC mutations61.
Clearly, driver mutations in KRAS, TP53, SMAD4 and/or
PIK3CA cooperate with APC mutations to drive the step-
wise progression from adenoma to carcinoma60,62,63.
Besides alterations in WNT, epidermal growth factor
(EGF), transforming growth factor-​β (TGFβ) or PI3K
signalling pathways that each provide a selective growth
advantage, these CRCs are characterized by chromosomal
instability (CIN)60. Although this conventional sequence of
mutational events explains the genetic basis of a size­able
fraction of CRCs, a much larger collection of mutated
genes were revealed with the advances of cancer genome
sequencing, thereby disclosing the heterogeneous nature
of CRC64,65.
Through a large-​scale, international effort, patients
with CRC (n = 3,962) were subclassified recently into
four consensus molecular subtypes (CMSs), each with
distinguishing biological characteristics: CMS1 (14%),
hypermutated subgroup with microsatellite instability
(MSI) and strong immune activation; CMS2 (37%),
canonical epithelial subgroup with marked WNT and
MYC activation; CMS3 (13%), epithelial subgroup with
evident metabolic dysregulation; and CMS4 (23%), dis-
playing prominent TGFβ activation, stromal invasion

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Microsatellite instability
(MSI). A state of hypermutation
due to impaired mismatch
repair of the genome. Most
common in colorectal, gastric
and endometrial cancer.
Mismatch repair
(MMR). A DNA repair pathway
that repairs DNA replication
errors, such as base–base
mismatches or erroneous
insertions or deletions.
Sessile serrated adenoma
(SSA). Pre-​malignant
flat (or sessile) polyps
predominantly observed
on the right side of the colon.
These polyps comprise the
main precursor lesion of
serrated carcinogenesis.
Left-​sided CRC
(Left-​sided colorectal cancer).
Tumours originating distal to
the splenic flexure (descending
colon, sigmoid colon and
rectum). Typically associated
with higher levels of WNT
signalling.
Right-​sided CRC
(Right-​sided colorectal cancer).
Tumours originating proximal
to the splenic flexure (caecum,
ascending colon and transverse
colon). Typically associated
with lower levels of WNT
signalling.
Loss of heterozygosity
(LOH). A frequent event in
cancer, involving loss of one
allele of a gene in the tumour
genome, after which a single
allele of the (mutated) gene
remains.
Polyploid
A condition in which a cell
contains more than two
complete sets of
chromosomes.
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and angiogenesis66. Strikingly, WNT pathway mutations
in CRC segregate to different CMSs (Fig. 2b). Although
APC mutations are prevalent in CMS2 and coincide with
CIN, RNF43 and ZNRF3 mutations as well as RSPO
family member gene fusions are found strongly enriched
within MSI-​linked hypermutated tumours of the CMS1
subgroup52,67,68. Thus, a relationship exists between the
type of WNT pathway mutation and the biological
context of the tumour.
In particular, RNF43 mutations and, to a lesser extent,
ZNRF3 mutations associate with the serrated pathway of
CRC development that originates from pre-​malignant
serrated polyps enriched for activating BRAF muta-
tions and mismatch repair (MMR) gene mutations
(mainly mutL homologue 1 (MLH1))67,69,70. Contrary
to APC mutations, the acquisition of RNF43 mutations
is considered a late event that drives the progression of
sessile serrated adenoma (SSA) to carcinoma67,71. Notably,
unlike APC-​mutant tumours, BRAF-​mutant, MSI-​linked
tumours do not display prominent nuclear β-​catenin
accumulation72. Thus, these findings suggest that RNF43
mutations generally associate with lower levels of WNT
pathway activation when compared with APC muta-
tions. Recent work showed that RNF43 mutations are
also enriched in signet-​ring cell carcinoma, a rare type
of early-​onset CRC that displays aggressive behaviour
and associates with poor prognosis73. Distinct from
APC-​mutant tumours, these signet-​ring cell tumours
do not display prominent activation of EGF or PI3K
pathways and only show low expression levels of conven-
tional WNT target genes, further emphasizing a distinct
pathogenic mechanism73.
Strikingly, APC and RNF43 mutations also associate
with distinct primary tumour locations within the
colon. Whereas APC mutations are predominantly
found in left-​sided CRC tumours, RNF43 mutations are
enriched in right-​sided CRC tumours65,74,75. In line with
the RNF43-​mutant profiles described above, right-​sided
tumours commonly harbour BRAF mutations and
display MSI-​linked hypermutation. Yet, even when
correc­ted for MSI status, RNF43 mutations correlate
with right-​sided CRC tumours, further supporting a
regional preference for WNT pathway alterations74. The
mole­cular basis for this preference remains unknown,
although a possible link may be the differential require-
ment for oncogenic WNT signalling levels that are
Table 1 | Molecular and clinical features of APC-​mutant and RNF43-​mutant colorectal cancer
Feature APC-​mutant CRC
Histological classification Tubular adenoma
Genetic subtype CIN, MSS
CMS Enriched in CMS2
Sidedness Enriched in left-​sided CRC
Co-​current mutations KRAS, TP53
Order of events Early and rate-​limiting driver event in
traditional adenoma sequence
Familial disorders FAP
APC, adenomatous polyposis coli; CIN, chromosomal instability; CMS, consensus molecular subtype; CRC, colorectal cancer; FAP,
familial adenomatous polyposis; MSI-​H, microsatellite instability high; MSS, microsatellite stable; RNF43, ring finger 43; SSA, sessile
serrated adenoma; ZNRF3, zinc and ring finger 3.
Reviews
usually higher in left-​sided CRC tumours76,77. Indeed,
physiological WNT signalling levels also display regional
differences along the healthy intestinal tract77, which
might be related to the developmental history of the
adult colon, with left and right regions originating from
the embryonic hindgut and midgut, respectively78,79.
Importantly, the primary tumour site differentially cor-
responds with prognosis. Left-​sided tumours mostly
metastasize to the liver and lung, for which resection and
chemotherapy is a potential cure, whereas right-​sided
tumours give rise to peritoneal metastases that remain
difficult to treat and lead to poor survival65,80,81.
In summary, driver mutations in APC and RNF43
are representative of CRC subsets with distinct charac-
teristics. An overview of the biological and molecular
characteristics of APC-​mutant and RNF43-​mutant CRC
subsets is provided in Table 1.
Hepatocellular carcinoma. Hepatocellular carcinoma
(HCC) is the sixth most frequent cancer worldwide
and the third leading cause of cancer-related death82.
HCC accounts for around 90% of patients with liver
cancer and is characterized by frequent overactiva-
tion of WNT–β-​catenin signalling. The most promi-
nent genetic alterations in the WNT pathway in HCC
comprise activating mutations in CTNNB1 (28–40%,
34/123 and 18/45) and LOF mutations in AXIN1 (11%,
13/123 and 5/45)83,84. In line with their tumour sup-
pressor role, AXIN1 mutations frequently asso­ciate
with loss of heterozygosity (LOH)48. As a large fraction
of human hepatocytes are polyploid, which may pro-
tect these cells from tumour suppressor loss85, it is
tempting to speculate that AXIN1 mutations are likely
acquired in a diploid state. Contrary to APC mutations
in CRC, CTNNB1 and AXIN1 mutations are usually
not present in pre-​malignant lesions of the liver but
instead associate with later stages of tumour develop-
ment48,86,87. Concordantly, WNT pathway activation
becomes apparent only in the late stages of HCC and is
thus considered a late step in the progression towards
hepatocarcinogenesis88.
Strikingly, CTNNB1 and AXIN1 mutations asso­
ciate with distinct HCC subtypes that display divergent
clinical and pathological features. CTNNB1 mutations,
directly mediating stabilization and nuclear accumula-
tion of β-​catenin, associate with a ‘non-​proliferative’ class
Refs RNF43-​mutant CRC Refs
93
Serrated adenoma 69,70
65,192
Mainly MSI-​H 52,67
66
Enriched in CMS1 66
64,65
Enriched in right-​sided CRC 65,74,75
60,62–64
BRAF, ZNRF3 54,67,69
60,62
Later event in serrated 52,67,193
carcinogenesis
46,55–57
Familial SSA 67,194,195
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Hypomorphic
of HCC, encompassing chromosomally stable tumours
A mutation resulting in that exhibit markers of differentiation and tend to retain
partial loss of function hepatocyte-​like markers89. These tumours express a clas-
of a gene, reducing but sical, canonical WNT activation gene expression signa-
not abolishing protein
ture and generally associate with a better prognosis83.
function.
By contrast, AXIN1 mutations are confined to a ‘prolife­
rative’ class of clinically aggressive HCC tumours, hall-
marked by poor differentiation, overexpression of cell
cycle markers and CIN89. In addition, AXIN1-​mutant
HCC tumours show activation of a diffe­rent tran­
scriptional programme in comparison with CTNNB1
mutations83,84,90.
Taken together, although mutations in CTNNB1 and
AXIN1 both converge on the inhibition of β-​catenin
turnover, their functional roles in liver carcinogenesis
are markedly different. The molecular basis for this
difference remains unresolved.
Molecular mechanisms of mutations
In the traditional view, mutation-​induced biallelic inacti-
vation of tumour suppressors induces the inappropriate
activation of signalling pathways that drive cell survival
and proliferation91. Accordingly, tumour suppressor
roles are typically studied using knockout approaches.
However, with the advances of cancer genome sequenc-
ing in human patients, a far more complex picture has
emerged. Mutations in tumour suppressor genes are
highly heterogeneous, giving rise to a range of errone-
ous proteins with unknown activity that are not captured
well by knockout models. In addition, this heterogeneity
poses a challenge to unambiguously distinguish passen-
ger mutations from driver mutations and to under-
stand the underlying cancer-​promoting mechanisms.
Moreover, besides loss of their growth suppressive ability,
mutant tumour suppressors may acquire novel functions
that promote tumorigenesis92. Herein, we discuss recent
insights into how patient-​derived mutations alter WNT
pathway tumour suppressor function in cancer.
Destruction complex mutations
APC truncating mutations in CRC generate a ‘just-​right’
level of WNT signalling. The tumour suppressor acti­
vity of APC primarily depends on its interactions with
β-​catenin and AXIN through multiple, short repeat
sequences located in the carboxy-​terminal half of its
long, intrinsically disordered region (Box 1). Although
CRCs usually carry biallelic APC mutations, APC func-
tion is typically not fully inactivated. In the vast majority
of cases, the 5′ half of the gene is hit by nonsense or
frameshift mutations that mediate expression of prema-
turely truncated APC protein variants (Fig. 2c). Notably,
mutations affecting both APC alleles are interdepend-
ent. In patients with FAP, germline mutations removing
all β-​catenin-​binding 20 amino acid repeats (20Rs) are
followed by somatic second-​hit mutations that retain
at least one 20R93–95. Conversely, when the germline
variant retains one or more 20Rs, the second hit will
lose all 20Rs, either by upstream truncation or by allelic
loss93–95. These findings have led to the ‘just-​right’ signal­
ling model, in which both APC alleles are selected to
retain sufficient β-​catenin regulatory activity to generate
an optimal window of signalling for tumour growth94.
10 | January 2021 | volume 21
In agreement with this assumption, progressive loss of
APC regulatory repeat sequences due to incremental
shortening of the protein corresponds with a stepwise
increase in WNT signalling in mouse embryonic stem
(ES) cells, human CRC cells and Drosophila embryos95–98.
Various lines of evidence indicate that APC variants
truncated within the ‘mutation cluster region’ (MCR),
ranging from codon 1286 to 1581, are best suited to
generate a defined level of WNT signalling that is most
optimal for tumour growth. The presence of monoal-
lelic or biallelic APC MCR truncations associate with
the most severe forms of polyposis in patients with FAP,
whereas truncating mutations shifted towards the amino
terminus or C terminus correspond with more attenu-
ated forms59,99. Furthermore, in sporadic CRC, 76% of
APC-​mutant cases carry at least one allele comprising
a MCR truncation76,100. Finally, preclinical mouse mod-
els of FAP also revealed that the most severe intesti-
nal polyposis occurs in heterozygous MCR-​truncated
Apc1322T/+ mice, followed by the commonly used APC
multiple intestinal neoplasia (ApcMin/+) mouse that car-
ries a shorter form, truncated at codon 850 (refs101–104).
Attenuated polyposis forms were observed for longer,
hypomorphic APC truncations at codon 1572 or 1638 that
retain more β-​catenin regulatory domains105,106 (Fig. 3a).
Notably, in these mouse models, polyposis primarily
develops in the small intestine, whereas in patients with
FAP and sporadic CRC the large intestine is the pre-
ferred site for polyp formation. The molecular basis for
this difference remains to be resolved but may relate to
differences in physiological WNT gradients between the
human and mouse intestinal tract77.
Which molecular features of MCR-truncated
APC allow for a specific level of β-​catenin-​mediated
trans­cription? First, loss of all three AXIN-binding
Ser-​Ala-Met-​Pro (SAMP) repeats appears to be a key
determinant for inactivation of the β-​catenin destruction
complex97,107. Despite lacking these conventional AXIN
binding sites, SAMP-​depleted APC truncations still form
a complex with AXIN, likely by employing an alternative
binding site located within the Armadillo repeat (ARM)
domain97,98,108–110 (Box 1). A second necessity is retention
of up to three β-​catenin-​binding 20Rs, through combined
biallelic APC truncations95,97. Although these remaining
20Rs still promote β-​catenin phosphorylation, they do
not facilitate β-​catenin destruction in APC-​depleted
cells108. Instead, these 20Rs are thought to confer residual
regulatory activity by mediating cytoplasmic β-​catenin
retention97. Finally, loss of the conserved catenin inhibi­
tory domain (CID; or ‘sequence B’), located adjacent to
the second 20R (20R2) (Box 1), is an additional require-
ment for driving high basal levels of WNT pathway activ­
ity, as shown in intestinal organoids98. The underlying
mechanism needs better characterization but seems to
involve a loss of interaction with α-​catenin, which pre-
vents stable interactions of APC with phosphorylated
β-​catenin111. In addition, diminished binding of the E3
ligase β-​TrCP by CID-​deleted APC might further impede
β-​catenin ubiquitylation and proteolysis98,112. Moreover,
CID removal may promote recruitment of the enzyme
ubiquitin-​specific processing protease 7 (USP7), which
further enhances β-​catenin stability by de-​ubiquitylation98.
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 Reviews

Box 1 | AXIN1-mediated and APC-​mediated organization of the β-​catenin destruction complex

The β-​catenin destruction complex is a central node of WNT signalling regulation27. The main activity of the complex is the
capturing and phosphorylation of β-​catenin, leaving a ‘recognition tag’ for subsequent ubiquitylation and proteasomal
degradation. The main organizer of the complex is the AXIN1 scaffold protein that interacts with all core components
of the complex and coordinates the activity of the kinases casein kinase 1 (CK1) and glycogen synthase kinase 3β (GSK3β).
AXIN1 employs its amino-​terminal RGS domain to interact with adenomatous polyposis coli (APC)10,23,199,200 and carries short
binding motifs for CK1 and GSK3β in its centrally located intrinsically disordered region10,26,201. The DIX domain located at
the AXIN1 carboxy terminus mediates self-​polymerization and nucleates the assembly of highly dynamic, spherically shaped
cytosolic puncta110,202,203. The role of APC is less well understood and primarily depends on its interactions with β-​catenin
and AXIN1 through multiple, short repeat sequences located in its long, intrinsically disordered region. For β-​catenin binding,
APC employs four clustered 15 amino acid repeat motifs (15Rs) and seven more dispersed 20 amino acid repeat motifs (20Rs)
that gain affinity for β-​catenin upon phosphorylation22,204. The second 20R (20R2) does not bind β-​catenin but cooperates
with an adjacent region called the catenin inhibitory domain (CID; or ‘sequence B’) to perform an essential but incompletely
understood step in the turnover of phospho-​β-​catenin95,97,110,111. Key interactions between APC and the AXIN1 RGS domain
are mediated by three Ser-​Ala-​Met-​Pro (SAMP) repeat motifs interspersed with 20R4 to 20R7 (refs199,201,205). A second, more
dynamic interaction with AXIN1 involves the APC N-​terminal Armadillo repeat (ARM) domain that interacts with the central
intrinsically disordered region of AXIN1 in a phosphorylation-​dependent manner110. The N terminus of APC carries
oligomerization activity206,207. The multimerizing properties of both AXIN1 and APC have led to the view that destruction
complex activity depends on the assembly of supermolecular non-​membrane-​bound compartments, often referred to as
biomolecular condensates143,208.
The figure shows the functional protein domains of APC, its close homologue APC2 as well as AXIN1 and AXIN2: ARM
mediates interaction with the cytoskeleton, protein phosphatase 2A (PP2A) and AXIN; 15R is for β-catenin binding; 20R is for
β-​catenin binding; CID is essential for promoting β-​catenin ubiquitylation; SAMP mediates AXIN interaction; basic region
mediates microtubule binding; end-​binding protein 1 (EB1) binding motif; mutation cluster region (MCR) of APC, which is
frequently hit by truncating mutations in human cancer; Tankyrase binding motif (TNKS); RGS mediates APC interaction;
and DIX mediates self-​polymerization. Binding motifs for GSK3β, β-catenin, Dishevelled (DVL) and CK1 are indicated.

AXIN1 or AXIN2 β-Catenin MCR β-Catenin

Oligomerization 15R 20R Basic region EB1
APC ARM
CID SAMP-AXIN1 or AXIN2
0 400 800 1,200 1,600 2,000 2,400 2,843
amino
acids
AXIN1 or AXIN2 β-Catenin
Oligomerization 20R Basic region EB1
APC2 ARM
CID SAMP-AXIN1 or AXIN2
0 400 800 1,200 1,600 2,000 2,303
amino
acids

APC AXIN1 or AXIN2, DVL

GSK3β β-Catenin CK1
AXIN1 TNKS RGS DIX

0 200 400 600 862

amino
acids

APC AXIN1 or AXIN2, DVL

GSK3β β-Catenin CK1
AXIN2 TNKS RGS DIX

0 200 400 600 843

amino
acids

Thus, MCR-​truncated APC balances loss of β-​catenin
degradation with residual regulatory activity to generate
an optimal dosage of β-​catenin-​mediated transcription to
drive tumour growth.
Relevance of less prevalent truncated APC forms. APC
truncations downstream of MCR have milder effects on
WNT signalling, occur at a lower frequency in human
cancer and are less well studied. However, several lines of
evidence indicate that these variants also perform tumo­
rigenic driver roles. Mice expressing hypomorphic APC
forms truncated after the CID display attenuated intes-
tinal polyposis but acquire multiple tumours in extra-​
intestinal tissues instead (for example, in the breast, liver
and bone)106,113 (Fig. 3a). In agreement, the fraction of
CID-​retaining APC truncations within TCGA database
is lowest in CRC (around 10% of 557 APC-​mutant cases)
but increases for other malignancies, such as stomach
adenocarcinoma (40% of 33) and uterine endometrioid
carcinoma (40% of 58), indicating that these cancer types
might select for lower WNT signalling levels (Fig. 3b,c).
Notably, residual suppressor activity of mutant APC is

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Reviews

a Phenotype Refs
Multiple intestinal adenomas 101,102
Min
Severe intestinal adenomas 103,104

lower nuclear β-catenin

1322T
Extra-intestinal adenomas 106
1572T
Multiple intestinal adenomas 105,107

destabilized APC protein

1638N

MCR
Oligomerization 15R 20R Basic region EB1
APC ARM
CID SAMP
0 400 800 1,200 1,600 2,000 2,400 2,843
amino
99% sporadic CRC, acids
both alleles <1,581
76% sporadic CRC,
at least one allele >1,286

b 20R1–3 CID SAMP1 c

Uterine Uterine
endometrioid endometrioid
carcinoma carcinoma
Stomach Stomach
adenocarcinoma adenocarcinoma
Skin cutaneous Skin cutaneous
melanoma melanoma
Bladder Bladder
urothelial urothelial
carcinoma carcinoma CID lost
Colorectal Colorectal CID
adenocarcinoma adenocarcinoma retained
0 1,000 2,000 3,000 0 0.2 0.4 0.6 0.8 1.0
Position of APC truncations Fraction of APC truncations

Fig. 3 | Correlation of APC truncation position and CID domain loss with tumour phenotype. a | Top: truncating
adenomatous polyposis coli (APC) mutation positions and corresponding tumour phenotypes in mouse models. Truncating
APC mutations are plotted against APC protein structure101–106. Bottom: the mutation cluster region (MCR) covers the
majority of APC mutations found in human colorectal cancer (CRC)76. b | Amino acid position of truncating (nonsense and
frameshift) mutations in APC shown for five cancer types with >5% frequency of APC mutations197. Positions of the first three
β-​catenin-​binding 20 amino acid repeats (20Rs) are indicated in purple. The first AXIN1 and AXIN2 binding Ser-​Ala-​Met-​Pro
(SAMP) repeat is indicated in pink. This repeat is lost in 99% of CRCs. The catenin inhibitory domain (CID) is indicated in
green. Medians are indicated by a red line. c | Truncating APC mutations in The Cancer Genome Atlas (TCGA) cancer types
from part b plotted for the fraction of mutations that retain or lose an intact CID. ARM, Armadillo repeat; EB1, end-​binding
protein 1; 15R, 15 amino acid repeat motif.

concentration-​dependent and may be further modulated
by alterations in protein stability105. In line with this,
a partial reduction in APC protein levels can sensitize
colon cells to WNT stimulation109. Furthermore, cooper-
ation with driver mutations of other pathways may alter
WNT dosage effects and tissue preference, as shown for
hypomorphic Apc1572T mice that acquire a prominent
intestinal polyposis phenotype upon co-​introduction
of SMAD4 mutations106. Lastly, it is noteworthy that
APC mutations may activate alternative driver path-
ways in cancer, such as Hippo–Yes-​associated protein
(YAP) signalling and CIN114,115. Although these events
are outside the scope of this Review, they evidently will
contribute to APC mutation-​driven cancer development.
Role of APC2 activity in APC-​mutant cancer. The
severity of APC mutations is balanced by expression
of its functional homologue APC2 that displays a
similar structural organization to APC but lacks the
β-​catenin-​binding 15 amino acid repeat (15R) region116,117
(Box 1). Whereas APC2 itself displays weak β-​catenin
down­regu­lating activity, its efficiency is enhanced by
heterodimerization with APC116–118. In fact, APC2 bind-
ing to MCR-​truncated APC promotes residual levels of
β-​catenin degradation in CRC cells118. Although this
activity cannot fully compensate for the loss of APC
function in the colon 117, a large fraction of CRCs
(43–95%, 29/66 and 97/102) display APC2 promoter
hypermethylation, indicating that higher levels of
β-​catenin activity acquired by loss of APC2 expression
might provide a growth advantage119,120.
The APC2-​dependent regulation of the WNT path-
way displays tissue specificity. For instance, deletion of
either Apc or Apc2 from the mouse mammary epithe-
lium did not affect tissue homeostasis, whereas simulta-
neous deletion of Apc and Apc2 led to excessive levels of

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stabilized β-​catenin and mammary tumour formation121.
Thus, in contrast to the colon, APC2 appears to com-
pensate for APC loss in breast tissue. Whether and how
APC2 expression balances WNT signalling thresholds in
other tissues thus deserves further investigation.
Consequences of AXIN1 loss of function. The mutational
spectrum of AXIN1 across all cancer types shows that
deep deletion occurs in 24% (57/240) of cases (Fig. 2c).
Owing to its key organizing role in destruction com-
plex assembly (Box 1), loss of AXIN1 is envisioned to
induce WNT-​independent β-​catenin activation in cells.
Indeed, Axin deletion corresponds with prominent
WNT-​mediated phenotypes in Caenorhabditis elegans
and Drosophila and induces embryonic lethality in
mice122–124. To study the potential cancer driver role of
AXIN1 loss in somatic cells, mouse models for condi-
tional disruption of Axin1 expression were developed
and, owing to the prevalence of AXIN1 mutations in
HCC83, evaluated for the effects on liver homeosta-
sis90,125. Loss of Axin1 in the adult liver induced an acute
increase in hepatocyte proliferation and, at 12 months
of age, 40–55% (27/67 and 5/9) of mice developed liver
tumours that histologically resemble human HCC90,125.
Contrary to Apc deletion or activating Ctnnb1 muta-
tions, Axin1-​deleted hepatocytes and tumours displayed
no or only a weak increase in the expression of con-
ventional WNT target genes125,126. However, the Axin1
loss-​induced gene signature showed strong similarity
to the poor-​prognosis ‘proliferative’ subclass of HCC
in which AXIN1 mutations are commonly found83,90.
Characteristic features comprised enhanced levels of
Notch and YAP signalling and a strong induction of fetal
gene expression90.
An evaluation of the consequences of AXIN1 loss
in other cell types and tissues is limited. A CRISPR
knockout screen performed in human small intestinal
organoids under conditions of WNT and RSPO with-
drawal promoted selective outgrowth of organoids
depleted of either APC or AXIN1 (ref.127). Furthermore,
long-​t erm treatment of a WNT-​h ypersensitive,
RSPO3-​overexpressing CRC line with potent WNT
secretion inhibitors induced outgrowth of a resistant
clone that acquired biallelic frameshift deletions in
AXIN1 (ref.128). Thus, these results confirm that AXIN1
loss mediates increased levels of WNT signalling in
intestinal organoids. In addition, induction of the nega­
tive feedback regulator AXIN2 was apparently not
sufficient to counteract the overall increase in WNT
signalling in these cells128.
Lastly, whereas AXIN1 mutations are detected in
only a small fraction of CRCs (3%, 19/549)64, depletion
of AXIN1 might occur through alternative mechanisms.
For instance, transcriptional upregulation of the E3
ubiquitin ligase RNF146 promotes CRC proliferation
and survival via ubiquitylation-​mediated degradation
of AXIN1 and activation of β-​catenin-​mediated tran­
s­cription129. Overexpression of RNF146 occurs in 6%
(13/205) of CRC cases and correlates with poor pro­g­
nosis129. Similarly, RNF146 overexpression was identi-
fied to drive proliferation and invasion via enhanced
WNT–β-​c atenin signalling in a large fraction of
naTure RevIeWs | CANceR
Reviews
NSCLCs130. As RNF146 is capable of degrading both
AXIN paralogues, upregulation of this E3 ligase might
be more potent than individual mutations in AXIN1
or AXIN2.
Consequences of AXIN2 mutation. Although AXIN2-​
mediated negative feedback appears insufficient to fully
compensate for AXIN1 loss125,128, somatic mutations in
AXIN2 are prevalent in various cancer types (Fig. 2a).
In addition, human germline mutations in AXIN2 are
associated with predisposition to lung cancer131,132, breast
cancer133,134 and CRC133,135–137. Evaluation of the mode of
action by which AXIN2 variants drive tumorigenesis is
limited. In one example, a recurring truncated AXIN2
mutant showed partial LOF upon ectopic expression
in cell lines138. Given the common co-​occurrence of
AXIN2 mutations with other WNT pathway mutations
in cancer, weak pathway activation by AXIN2 LOF may
act synergistically with these mutations to increase
signalling levels or provide drug resistance138–140. Further
characterization of the molecular mechanisms of AXIN2
mutations and their cooperativity with other genetic
alterations is required to gain a better understanding
of how these mutations contribute to the initiation and
progression of cancer growth.
Mechanisms of AXIN1 missense mutations: beyond loss
of function. Despite the useful knowledge gained from
studying AXIN1 deletion, the largest fraction of genetic
alterations of AXIN1 in human cancer comprise mis-
sense mutations (Fig. 2c) that are found scattered over
the entire length of the gene, complicating functional
predictions. In-​depth analysis of missense mutations
in the N-​terminal RGS domain recently revealed that
the ensuing mutant AXIN1 proteins may acquire novel
functional properties to drive tumour growth (Box 1).
Mechanistically, missense driver mutations were found
to structurally destabilize the RGS domain and medi-
ate formation of small-​scale aggregates that rewire the
AXIN1 interactome to promote basal WNT pathway
activation141 (Fig. 4). Furthermore, insertion of mis-
sense RGS-​mutant AXIN1 in the endogenous locus
promoted tumour growth in vivo in Drosophila, con-
firming the cancer driver activity. The role of aggrega-
tion as the underlying mechanism was validated by the
introduction of secondary mutations that suppressed
aggregation and rescued tumorigenic effects141. Thus,
these findings indicate that tumour suppressor mis-
sense mutations may contribute to cancer growth via
unforeseen mechanisms.
However, the relevance of most other missense muta­
tions remains unknown. Owing to the role of AXIN1 as
a hub protein as well as its ability to self-​polymerize142,
multiple modes of interference with these functions
by mutations can be envisioned. Mechanistic insight
will help classify the large number of reported muta-
tions according to their mode of action. In addition,
as destruction complex activity depends on the abso-
lute levels and ratio of APC and AXIN1 proteins143,
evaluation of molecular and cellular consequences
of mutant AXIN1 at the endogenous level will be
essential.
volume 21 | January 2021 | 13
Reviews

Wild-type AXIN1
Puncta In human cancer, the mutational spectrum of RNF43
is dominated by truncating and missense mutations,
whereas ZNRF3 is more frequently hit by missense
mutations and deletions (Fig. 2c). Functional analysis of
APC different RNF43 mutational classes indicates that LOF
β-TrCP can be achieved by various mechanisms (Fig. 5a–d). First,
AXIN1 RGS DIX mutations that truncate RNF43 upstream of the E3 ligase
CK1 β-Catenin GSK3β RING domain will cause either loss of protein expres-
P P P sion or expression of non-​functional protein (Fig. 5b).
A prominent example is the R117Afs*41 RNF43 muta-
tion that accounts for 8–12% (4/51 and 6/50) of RNF43
mutations in CRC and 4% (2/58) of RNF43 mutations in
endometrium cancer52. Second, missense mutations
in the extracellular domain, as reported in pancreatic
RGS missense mutant AXIN1 Nano-aggregates cancer145, can mediate RNF43 retention in the endo-
plasmic reticulum (ER), likely by misfolding and ER
quality control-​mediated retention and clearance149.
Owing to their inability to reach the plasma membrane,
AXIN1 DIX these mutants fail to downregulate WNT receptors.
CK1 GSK3β
RGS Furthermore, ER-​trapped RNF43 mutants may harbour
β-Catenin dominant negative activity and thus have increased
β-Catenin β-Catenin tumorigenic capacity compared with gene deletions149
(Fig. 5c). A third mechanism comprises depletion of
Fig. 4 | Tumorigenic mechanism of AXIN1 missense mutations. Top: wild-​type AXIN1 mutant RNF43 via nonsense-​m ediated mRNA decay
recruits and organizes the main components of the β-​catenin destruction complex to (NMD), which may be induced following the gain of
facilitate efficient β-​catenin degradation. AXIN1 self-​polymerizes via its DIX domain premature termination codons (PTC)150 (Fig. 5d). In sup-
to form large biomolecular condensates that are visible as spherically shaped cytosolic port of this, NMD inhibition stabilized the mRNA of
puncta in the cell. Immunofluorescent microscopy image shows wild-​type AXIN1 several trunca­ting RNF43 variants in CRC cell lines151.
overexpressed in HeLa cells. Bottom: cancer missense mutations (red stars) in the
However, the role of NMD has been debated, particu-
AXIN1 RGS domain disrupt its structure and promote the formation of small-​scale AXIN1
larly for the RNF43 G659Vfs*41 hotspot mutation that
aggregates that form a dysfunctional complex that fails to form puncta and displays
impaired capacity to degrade β-​catenin. Immunofluorescent microscopy image shows is highly prevalent in colorectal, endometrium and
AXIN1 RGS missense mutant overexpressed in HeLa cells. Fluorescently labelled AXIN1 stomach MSI-​high cancer subsets52,152. Because the
is shown in white, nucleus of the cell (DAPI staining) is shown in blue. APC, adenomatous RNF43 G659Vfs*41 protein appears fully functional
polyposis coli; β-​TrCP, β-​transducin repeat-​containing protein; CK1, casein kinase 1; in overexpression assays153,154, NMD-​mediated mRNA
GSK3β, glycogen synthase kinase 3β; P, phosphorylation. Immunofluorescence images depletion was proposed as a mode of action. Indeed,
are original, unpublished images from the authors’ examination of cells. patient-​derived or genetically engineered human colon
organoids harbouring RNF43 G659Vfs*41 combined
Mutations in WNT receptor regulators with ZNRF3 inactivation acquired RSPO-​independent
RNF43 and ZNRF3 loss-​of-​function mutations. Gene growth properties, suggesting LOF effects67,155. Also, in
deletion experiments in mice revealed that the tumour line with NMD susceptibility, RNF43 mRNA levels were
suppressor roles of RNF43 and ZNRF3 are redundant decreased in G659Vfs*41-bearing human tumours69.
in the intestine and that simultaneous inactivation of However, these findings were challenged recently
both genes is a requirement for tumour growth14,144. by a study in which RNF43 G659Vfs*41 mRNA was
In line with these findings, human serrated CRCs often found stably expressed. Moreover, deletion of this
present with mutated RNF43 and concomitant down- variant from CRC cell lines still induced an increase in
regulation of ZNRF3, either by mutations or epigenetic WNT–β-​catenin signalling, suggesting that LOF of this
mechanisms54. However, modelling RNF43 and ZNRF3 RNF43 mutant is incomplete154. Although the reason for
loss in various cells and tissues indicates context-​ these discrepancies remains unknown, the mutational
dependent effects, in which one of the paralogues might background and tissue context might play a role.
be dominant over the other. For instance, individual
deletion of mouse Rnf43 drives inappropriate WNT sig- An oncogenic role for RNF43 truncations in cancer.
nalling and hyperplastic growth in the pancreas145 and Accor­ding to the rules of NMD, nonsense or frameshift
stomach146, whereas Znrf3 loss drives tumour formation muta­tions that hit the centre of the large penultimate
Nonsense-​mediated mRNA in the adrenal cortex147. Notably, RNF43 LOF mutation-​ exon of RNF43 have a high probability to escape NMD150.
decay
bearing WNT-​hypersensitive CRC subsets frequently How these truncated RNF43 variants interfere with
(NMD). A molecular
mechanism of mRNA display epigenetic downregulation of additional WNT WNT signalling and tumorigenesis remains poorly
surveillance that recognizes pathway negative feedback regulators, including AXIN2, understood, as current knowledge largely relies on gene
and eliminates mRNAs as well as NOTUM and WNT inhibitory factor 1 (WIF1), knockout strategies. A recent study revealed a class of
harbouring premature that counteract the activity of WNTs148. Thus, such alter- truncated RNF43 variants that promotes tumorigenesis
termination codons, thus
contributing to the protection
ations may further enhance the duration and intensity by a mechanism distinct from LOF39. These RNF43
of eukaryote cells from of the WNT responses of RNF43- and ZNRF3-​mutant variants that have lost the distal half of their cytosolic
putative toxic proteins. cells and promote tumour progression. tail (Fig. 5a) fully retained their ability to downregulate

14 | January 2021 | volume 21 www.nature.com/nrc

 Reviews

WNT receptors yet induced WNT-​independent
β-​catenin-​mediated transcription (Fig. 5e). Loss of the
C terminus was found to interfere with a second suppres-
sor role of RNF43 that involves binding and regulating
the activity of the β-​catenin destruction complex. In par-
ticular, the kinase CK1 was identified as a dynami­cally
regulated interaction partner that phosphorylates the
RNF43 tail to facilitate its role in β-​catenin downregu­
lation (Figs 1b,5a). Upon truncation, the interaction of
RNF43 with the destruction complex is misregulated,
leading to trapping of AXIN1 and CK1 at the plasma
membrane and, at the same time, β-​catenin stabiliza-
tion and target gene transcription (Fig. 5e). Furthermore,
the introduction of RNF43 truncating mutations in

a
DIR CK1 Onco
RNF43 SP PA TM RING

0 200 400 600 783

amino
DIR acids
ZNRF3 SP PA TM RING

0 200 400 600 800 936

amino
acids
b Loss of functional domains c Dominant negative mislocalization

RNF43 FZD WNT LRP5 WNT

or LRP6

DVL DVL
Ubiquitination APC AXIN1 APC AXIN1
GSK3β Mutant GSK3β
CK1 RNF43 CK1
Wild-type RNF43
or ZNRF3
Hypersensitive Hypersensitive
WNT signalling WNT signalling

ER

d RNF43 loss of function e Oncogenic RNF43

WNT Oncogenic
RNF43

P P
DVL CK1 CK1 Ub
APC AXIN1 AXIN1AXIN1 Endosome Ub
NMD GSK3β Ub
CK1

Nonsense
RNF43 mRNA AXIN1/2
WNT- CK1 β-Catenin
Hypersensitive independent Lysosomal
WNT signalling signalling degradation APC GSK3β

Fig. 5 | RNF43 and ZNRF43 structural organization and tumorigenic mechanisms of RNF43 mutations. a | WNT target
genes ring finger 43 (RNF43) and zinc and ring finger 3 (ZNRF3) are essential regulators of stem cell homeostasis. The
encoded homologous transmembrane proteins act in a negative feedback loop by employing their intracellular E3-​ligase
catalytic RING domain to ubiquitylate and remove the receptors Frizzled (FZD) from the cell surface by endocytosis13,14.
The mode of RNF43- and ZNRF3-​mediated substrate recognition remains poorly characterized but might involve an
interaction of the centrally located Dishevelled-​interaction region (DIR) with the Dishevelled (DVL) protein that binds
FZD under WNT-​stimulated conditions198. More distal carboxy-​terminal interactions with casein kinase 1 (CK1) underlie
a second RNF43-​mediated suppressor role that facilitates β-​catenin degradation by the destruction complex. This role
is exploited by truncating cancer mutations in a defined region of the RNF43 C-​terminal tail to drive oncogenic WNT
signalling39. b | RNF43 truncations that remove functional intracellular domains lead to hypersensitive WNT signalling.
c | RNF43 missense mutations that mislocalize and are dominant negative over wild-​type RNF43 and ZNRF3 alleles drive
hypersensitive WNT signalling. d | RNF43 truncations that lead to loss of function by nonsense-​mediated mRNA decay
(NMD) mediate hypersensitive WNT signalling. e | Oncogenic RNF43 truncations that trap CK1 and AXIN1 at the plasma
membrane drive WNT-​independent β-​catenin signalling activation. APC, adenomatous polyposis coli; ER, endoplasmic
reticulum; GSK3β, glycogen synthase kinase 3β; LRP5, low-​density lipoprotein receptor-​related protein 5; Onco, region in
RNF43 that mediates oncogenic activity upon truncation (amino acids 504–563); P, phosphorylation; PA, protease-​associated
domain; SP, signal peptide; TM, transmembrane domain; Ub, ubiquitin.

naTure RevIeWs | CANceR volume 21 | January 2021 | 15

Reviews
primary human colon organoids induced activation of
a β-​catenin-​mediated growth programme in the absence
of ZNRF3 mutations, thus confirming their dominant
oncogenic activity39.
Unlike APC or RNF43 LOF mutations67,156,157, onco-
genic RNF43 mutations induced a state of senescence-​
like growth arrest, and their expression was tolerated
only in TP53-​null human colon organoids39. These find-
ings indicate that, like many conventional oncogenes158,
these truncated RNF43 variants induce oncogenic
stress and might represent a late event during tumori­
genesis. Indeed, patient-​derived tumours carrying
oncogenic RNF43 mutations harboured mutations in
senescence-​related pathways as well39. Another impor-
tant distinction from LOF mutations is that expression
of oncogenic RNF43 variants confers resistance to
anti-​WNT-​based therapy39. Thus, improved knowledge
of tumour-​promoting mechanisms of patient-​derived
RNF43 mutations will help to classify and predict
therapeutic efficacy, as discussed below.
Opportunities for precision medicine
The pivotal role of WNT–β-​catenin signalling alterations
in human cancer growth has attracted great interest in the
development of strategies for therapeutic targeting of this
pathway. These ambitions were further enforced by the
observation that, even at advanced stages of APC-​mutant
intestinal cancer in mice, restoration of APC expression
drives differentiation of tumour cells and restoration of
normal tissue homeostasis159. Despite the identification
of numerous WNT pathway antagonists as well as inhibi­
tory small molecules, none of these drugs has achieved
clinical approval. A major challenge for clinical targeting
is to inhibit WNT signalling in cancer cells while avoid-
ing on-target toxicity in healthy tissues owing to altera-
tions in adult stem cell homeostasis. For a comprehensive
evaluation of WNT pathway inhibitors that are currently
being evaluated in clinical trials, we refer the reader to
other recent reviews17,160. Below, we discuss how recent
advances in the understanding of WNT–β-​catenin path-
way tumour suppressor alterations in cancer cells revealed
druggable vulnerabilities and facilitated the discovery of
drugs with potential for the development of innovative
therapeutic approaches. In addition, we highlight how
accumulating insights into the mechanism of action of
patient-​d erived mutations improves stratification
of patients for applications of precision medicine.
Targeting destruction complex activity
Improving β-​catenin destruction complex activity in
cancer cells imposes a challenge, owing to the frequent
loss of key protein–protein interaction elements of its
main organizers, APC and AXIN. Yet recent progress in
understanding the molecular events that drive WNT sig-
nalling following such LOF events has revealed several
targetable vulnerabilities27.
Residual destruction complex activity observed in
tumours carrying APC truncations offers an opportu­nity
to enhance β-​catenin turnover in cancer cells. The small
molecule XAV939 was identified as a potent enhancer of
destruction complex activity by promoting AXIN stabili-
zation in APC-​mutant CRC cell lines161. Mechanistically,
16 | January 2021 | volume 21
XAV939 inhibits the poly(ADP-​ribosyl)ating (PARylating)
enzymes Tankyrase 1 (TNKS1) and TNKS2, which prevents
AXIN1 and AXIN2 PARylation as well as subsequent
RNF146-​mediated ubiquitylation and proteasomal
degradation161. Besides promoting AXIN stabilization,
XAV939 treatment enhances levels of TNKS1 and
TNKS2, which might further contribute to WNT path-
way suppression by a non-​catalytic scaffolding role, via
co-​polymerization with AXIN1 and AXIN2162. Despite
these promising findings, follow-​up studies investi-
gating the relationship between truncated APC length
and sensitivity to TNKS inhibitors revealed conflicting
results100,163,164. In particular, cell lines carrying APC
MCR truncations (retaining one or two 20Rs) displayed
resistance to TNKS inhibition in vitro, whereas tumours
carrying such APC mutations displayed greater sensi-
tivity to treatment in vivo in mouse models, in com-
parison with shorter APC truncations163,164. Potentially,
this discrepancy might be explained by differences in
expression levels of the negative regulators of the WNT
pathway AXIN2 and APC2, both of which are stabilized
upon TNKS inhibitor treatment139,165. Alternatively,
resistance to TNKS inhibition might be promoted by
co-​o ccurring oncogenic alterations in other cancer
pathways as shown for mutant KRAS166, although here
the underlying mechanism remains to be resolved.
A further concern is the observation that elevated doses
of single-​agent TNKS inhibitor treatment necessary for
tumour inhibition elicits intestinal toxicity, weight loss
and death in rodents163,167. Nevertheless, the application
of reduced doses of TNKS inhibitors in combination
treatment regimens revealed promising antitumour
effects163,167. Thus, elucidation of how cellular context
impacts on TNKS inhibitor effectiveness as well as their
value in combination with other targeted drugs will be
essential to determine optimal strategies for therapeutic
application.
Another potential vulnerability that emerges upon
APC truncation was recently revealed. In CRC cells
displaying loss of the APC CID domain, increased USP7-
mediated de-​ubiquitylation of β-​catenin was identi-
fied as a main driver of aberrant WNT signalling98.
Consequently, treatment with USP7 inhibitors sup-
pressed WNT activation in APC-​mutant CRC cell lines
and intestinal organoids and inhibited tumour growth
in vivo98,168,169. However, these findings were challenged
by a recent study in which USP7 inhibitors were instead
identified as positive regulators of WNT–β-​catenin
signalling, via inhibition of AXIN stabilization170. The
molecular basis for discrepancy between these studies
remains unclear and will require a careful comparison of
cellular systems and different classes of USP7 inhibitors
that were used.
In a screen for compounds that co-​regulate β-​catenin
and RAS protein stability, a small molecule was iden-
tified that enhances destruction complex activity. This
compound, called KYA1797, binds the AXIN1 RGS
domain in a surface located close to the APC binding
site171. By an incompletely understood mechanism,
KYA1797–AXIN1 binding increases GSK3β activity
and promotes both β-​catenin and RAS degradation171.
In line with these findings, follow-up work indicates that
www.nature.com/nrc

Dysgeusia
Distortion of the sense of taste.
naTure RevIeWs | CANceR
β-catenin and RAS interact directly and, furthermore,
degradation of β-​catenin precedes GSK3β-​dependent
RAS degradation 172. KYA1797 inhibits the growth
of CRCs that carry APC and KRAS mutations in
mice via the degradation of both RAS and β-catenin
oncogenes171,172.
Targeting WNT-​dependent cancers
The discovery that the RNF43 and ZNRF3 muta-
tions confer a state of WNT-​dependent growth has
opened up the possibility to identify cancer subsets
that are sensitive to treatment with WNT inhibitors
or receptor-​blocking antibodies13,14,145. A highly potent
approach to block autocrine and paracrine WNT signal-
ling is inhibition of the production of all active WNTs
by targeting Porcupine (PORCN), an ER-​resident
O-​acyltransferase that mediates WNT palmitoylation, an
essential step in WNT biosynthesis173. Indeed, preclini-
cal data showed that PORCN inhibitors effectively sup-
pressed WNT-​driven growth of engineered Rnf43- and
Znrf3-​mutant mouse intestinal tumour models as well
as patient-​derived RNF43-​mutant CRC and pancreatic
ductal adenocarcinoma (PDAC) organoids69,155,174. In an
effort to exploit this vulnerability, ongoing clinical trials
employ RNF43 mutations as a predictive biomarker for
treatment of patients with CRC and PDAC with PORCN
inhibitors175–180. Despite promising preclinical results,
on-​target adverse effects by PORCN inhibitors including
bone loss and dysgeusia were reported, likely limiting the
dosages that can be applied in the clinic181–183. However,
recent work indicates that PORCN inhibitors might be
used successfully at lower doses in drug combination
strategies, offering improved antitumour efficacy183.
In particular, dual treatment with PORCN and PI3K
inhibi­tors potently inhibited the growth of PDAC xeno­
grafts, owing to suppression of cell proliferation and
glucose metabolism184. Thus, evaluation of synergistic
effects of PORCN inhibitors with other targeted drugs
appears to be a valuable strategy that deserves explora-
tion for the treatment of WNT-​hypersensitive cancers.
Furthermore, based on recent insights, the efficacy of
PORCN inhibitors may be enhanced by improved strati­
fication of patients with RNF43 mutations. To achieve
this, discrepancies in the classification of RNF43 muta-
tions as passengers or drivers will need to be resolved.
Moreover, patients who carry RNF43 mutations that
promote WNT-​independent growth39, or other coexis­
ting mutations that predict resistance to PORCN
inhibition128,154, would need to be excluded. Finally, in
tissues where RNF43 and ZNRF3 display redundancy,
the status of ZNRF3 expression will help to confirm
WNT-​addicted tumours.
As PORCN inhibitors affect all WNT-​mediated path-
ways, more selective strategies are being developed to
mitigate on-​target toxicity. For example, a genome-​wide
CRISPR screen identified FZD5 as the primary WNT
receptor essential for viability in RNF43-​mutant PDAC
cell lines185. In agreement, FZD5 inhibitory antibodies
blocked the growth of RNF43-​mutant PDAC cell lines
in vitro and in vivo185. In addition, FZD5 targeting
inhibited survival of RNF43-​mutant CRC organoids,
whereas APC-​mutant organoids remained unaffected185.
Reviews
Finally, selective blockade of WNT3 binding to the LRP6
receptor by single-​chain antibodies inhibited hyper-
proliferation and promoted terminal differentiation of
Rnf43- and Znrf3-​mutant mouse intestinal organoids186.
Thus, more selective targeting of WNT signalling by
antagonizing specific receptors or WNTs holds pro­mise
to overcome some of the limitations associated with
PORCN inhibitor treatment.
Lastly, the spectrum of targetable WNT-​dependent
tumours might be broader then currently anticipated.
Unexpectedly, the growth of APC-​mutant human gas-
tric cancer tumours was inhibited in vitro and in vivo
by treatment with Vantictumab (OMP-18R5), an
inhibi­tor of several FZDs that has been evaluated in
phase Ib clinical trials for advanced pancreatic, lung
and breast cancer 187,188. The extent to which other
human cancer (sub)types carrying downstream WNT
pathway mutations may still depend on WNT recep-
tor activity for their survival has yet to be investigated.
Additionally, mutations outside the WNT pathway may
also yield WNT-​hypersensitive tumours, as indicated
for combined mutations in TP53 and CDH1 (encoding
E-​cadherin) in gastric cancer49. Thus, there might be an
unexplored subset of cancers that could benefit from
WNT inhibition.
Conclusions and perspectives
Knowledge of the molecular mechanisms underlying
tumorigenesis is a necessary step for the implementa-
tion of precision cancer medicine. Although model-
ling WNT tumour suppressor inactivation using gene
deletion approaches in cells and organisms has gener-
ated a wealth of information, these models only repre-
sent a limited part of the mutational spectra identified
in human cancer genomes (Fig. 2c). Moreover, current
prediction algorithms for driver mutations in WNT
pathway tumour suppressors are suboptimal owing to
the scattered distribution of mutations as well as the
presence of large, intrinsically disordered regions in
the encoded proteins189,190 (Box 1). As illustrated by the
examples given in this Review, a more comprehensive
experimental evaluation of the functional effects of
different classes of patient-​derived tumour suppressor
mutations is needed to distinguish driver mutations from
passenger mutations, define common mechanistic rules,
understand tissue specificity and improve guidance for
therapeutic decisions. Recent advances in genome edit-
ing and applications of organoids as faithful models of
tumour growth and metastasis are timely developments
that will be of great value to address these issues191.
An immediate challenge for successful application
of WNT-​blocking or receptor-​blocking agents is the
robust identification of WNT-​addicted cancer subsets.
Eligible patients may be stratified based on the presence
of confirmed RNF43 LOF mutations and, in specific
tissues, concomitant LOF of ZNRF3. In addition, a more
comprehensive search for additional predictive markers
that signify WNT-​d ependent growth, as reported
for combined TP53 and CDH1 mutations in gastric
cancer49, deserves further exploration and might help
to expand targetable patient subsets. Furthermore, an
in-​depth analysis and understanding of how epigenetic
volume 21 | January 2021 | 17
Reviews

silencing and transcriptional loss of negative feedback
regulators contribute to the development and progres-
sion of WNT-​driven cancers will be needed to iden-
tify and expand clinically actionable patient subsets.
Of equal importance is the need to explore mutations
and pathways that provide resistance to WNT-​blocking
or receptor-​blocking therapy. Besides a specific set of
truncating RNF43 mutations, downstream pathway
mutations in APC and AXIN1 were identified as poten-
tial escape routes, although such effects might depend
on mutation positioning and the tissue context39,128,154,188.
The identification of reliable markers may be accelerated
by in vitro drug screening using patient-​derived tumour
organoids.
Finally, although we focused on the molecular con-
sequences of individual WNT tumour suppressor muta-
tions, clearly the roles of mutational context, tumour
heterogeneity and interactions with other major cancer
pathways deserve further investigation. Hopefully, by
combining molecular insights with information coming
from early-​phase clinical trials, the efforts to target WNT
signalling will translate soon into effective therapies.
Published online 23 October 2020

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Acknowledgements
The authors thank members of the Maurice laboratory for
helpful discussions and suggestions, specifically I. Jordens
and A. Venhuizen for critically reading the manuscript and
A. Cristobal Gonzales de Durana for providing AXIN1
immuno­fluorescence images. This work is part of the Oncode
Institute, which is partly financed by the Dutch Cancer
Society. This work was supported by European Research
Council Starting Grant 242958, the Netherlands
Reviews
Organi­zation for Scientific Research NWO VICI Grant
91815604 and ZonMW TOP Grant 91218050 (to M.M.M.).
Author contributions
J.M.B., N.F. and M.M.M all researched data for the article,
provided substantial contributions to discussions of the con-
tent and contributed equally to writing the article and to the
review and/or editing of the manuscript before submission.
Competing interests
The authors declare no competing interests.
Peer review information
Nature Reviews Cancer thanks B. O. Williams, M. Waterman
and the other, anonymous, reviewer(s) for their contribution
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