Author's Accepted Manuscript

Functional Crosstalk Between WNT Signaling

and Tyrosine Kinase Signaling in Cancer
J.N. Anastas Phd

www.elsevier.de/endend

PII: S0093-7754(15)00190-6
DOI: http://dx.doi.org/10.1053/j.seminoncol.2015.09.020
Reference: YSONC51876

To appear in: Semin Oncol

Cite this article as: J.N. Anastas Phd, Functional Crosstalk Between WNT Signaling
and Tyrosine Kinase Signaling in Cancer, Semin Oncol, http://dx.doi.org/10.1053/j.
seminoncol.2015.09.020

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Functional Crosstalk between WNT Signaling and Tyrosine Kinase Signaling

in Cancer

J.N. Anastas, Phd1,2*

1) Harvard Medical School Department of Cell Biology, Boston, MA USA

2) Boston Children’s Hospital Division of Newborn Medicine, Boston, MA

USA

* To whom correspondence should be addressed:

Boston Children’s Hospital

Enders Building 926

300 Longwood Ave

Boston, MA 02115

jamie.anastas@childrens.harvard.edu

The author has no conflict of interest, financial or otherwise, to report.

1
ABSTRACT

Extensive molecular characterization of tumors has revealed that the activity of

multiple signaling pathways is often simultaneously dampened or enhanced in

cancer cells. Aberrant WNT signaling and tyrosine kinase signaling are two

pathways that are frequently up- or down-regulated in cancer. Although signaling

pathways regulated by WNTs, tyrosine kinases, and other factors are often

conceptualized as independent entities, the biological reality is likely much more

complex. Understanding the mechanisms of crosstalk between multiple signal

transduction networks is a key challenge for cancer researchers. The overall goals

of this review are to describe mechanisms of crosstalk between WNT and tyrosine

kinase pathways in cancer and to discuss how understanding intersections

between WNT and tyrosine kinase signaling networks might be exploited to

improve current therapies.

2
Signaling crosstalk in cancer

The molecular characterization of tumors reveals that multiple different signaling

pathways are often simultaneously up- or down-regulated in cancer cells.

Aberrant activity of tyrosine kinase-dependent signaling networks is commonly

observed in a wide variety of tumors as highlighted by recent genome wide

association studies cataloging driver mutations in human tumors revealing that

both receptor tyrosine kinases (RTKs) and non-receptor tyrosine kinases are

frequently mutated in cancer. Hyper-activation or hypo-activation of WNT

signaling pathways can similarly contribute to carcinogenesis in a context-

dependent manner. Simultaneous up- or down-regulation of multiple signaling

pathways has profound consequences on tumor cell biology. First, there can be

synergy between multiple signaling pathways resulting in more pronounced

changes in tumor cell behaviors such as proliferation, metastasis, or the

development of drug resistance. Second, simultaneous defects in multiple

pathways can lead to emergent biological properties that would not be observed if

only a single pathway were altered. Finally, multiple signaling pathways may act

in a redundant manner such that one pathway can compensate for the other when

only a single pathway is targeted therapeutically. This review will first summarize

current knowledge of the mechanisms of crosstalk between WNT and tyrosine

kinase pathways in cancer, and will go on to describe how increasing our

3
understanding of signaling crosstalk may be a key step towards the development

of personalized cancer therapies.

Context-dependent roles for WNT signaling in cancer

A role for the WNT/β-catenin signaling in cancer was first described over three

decades ago in mouse models of mammary cancer where aberrant overexpression

of WNT1 induces spontaneous mammary tumorigenesis.1-3 Additional studies

revealed that inherited mutations in Adenomatosis Polyposis Coli (APC), which

acts as a negative regulator of WNT signaling, predisposed patients with Familial

Adenomatous Polyposis (FAP) to develop colorectal tumors reviewed in:4,5. Since

these initial findings showing that hyper-activation of the WNT signaling pathway

promotes tumorigenesis in the breast and colon, we have developed a more

nuanced understanding of the role of WNT signaling in cancer. First, multiple

studies reveal that WNTs do not exclusively signal through β-catenin, but can

elicit complex downstream responses involving networks of effector proteins such

as small GTPases, transcription factor networks and kinase-dependent signaling

cascades. Second, it is now clear that aberrant WNT signaling does not always

promote tumor development, but both positively and negatively regulate nearly all

aspects of carcinogenesis ranging from tumor initiation, to the development of

metastatic lesions, to the development of therapeutic resistance in aggressive

tumor types.6

4
β-catenin-dependent and β-catenin–independent WNT signal transduction

networks

WNTs are a family of 19 secreted glycoproteins7 that can regulate diverse cell

behaviors. WNTs act at the cell surface where they can bind several different

classes of receptors including the ten members of the FZD family of G-protein

coupled receptors (GPCRs) as well as receptor tyrosine kinase family receptors

RYK, ROR1,2 and PTK7.8 Some WNTs regulate changes in cell behavior

through a signaling network ultimately resulting in the stabilization of β-catenin

and the activation of β-catenin -dependent transcription. In the absence of WNT

stimulation, a destruction complex containing the proteins APC, GSK3β, CKII,

and AXIN, phosphorylates and targets β-catenin for degradation by the

proteasome (Figure 1A). The binding of a subset of WNTs, such as WNT3A and

WNT1, to FZD and LRP5/6 co-receptors transduces a signal across the plasma

membrane resulting in the activation of DVL which acts to inhibit the destruction

complex resulting in the stabilization of β-catenin. Stabilized β-catenin can then

enter the nucleus where it can regulate the transcription of multiple target genes in

cooperation with the TCF/LEF family of transcription factors (Figure 1B).

Other WNTs such as WNT5A and WNT11 similarly interact with RYK,

ROR, and the FZD family of transmembrane receptors and require the activity of

DVL, yet do not regulate β-catenin -dependent transcription. Instead, these β-

5
catenin -independent WNTs regulate the activity of a variety of downstream

signaling molecules in a context-dependent manner. In some biological contexts,

β-catenin-independent WNTs can induce a calcium flux resulting in the activation

of Ca2+-dependent signaling pathways such as the PKC, and CAMKII cascades.

β-catenin-independent WNTs can also promote the activation of a variety of other

kinase cascades such as the JNK/AP-1 pathway and the MEK/ERK pathway.

Finally, β-catenin-independent WNTs are frequently linked to cell migration and

motility, and the binding of these WNTs to members of the FZD family of

receptors can also alter the activity of small GTPases involved in regulating

cytoskeletal dynamics such as, RHOA, RAC1 and CDC42.

β-catenin-independent WNTs can also signal via the planar cell polarity

(PCP) pathway, which is critically important for many biological processes in

vertebrates such as the promotion of gastrulation movements during the

development and maintenance of tissue polarity.9-13 Although LRP5/6 co-

receptors have not been linked to PCP phenotypes, disrupting the function of FZD

receptors,13-19 as well as of the RTK family of WNT receptors ROR2 and RYK,

leads to PCP phenotypes,10,13,20,21 suggesting that WNTs may regulate PCP

through multiple different receptors. Other transmembrane proteins such as

VANGL,22-24 PTK725,26 and CELSR,27-29 genetically or biochemically interact

with WNTs and FZDs to regulate PCP possibly by acting as co-receptor

molecules. Downstream of the WNT-receptor complexes, PCP signaling requires

6
the intact function of DVL,15,16,19,30 as well as a number of other cytosolic factors

including the PDZ-domain containing proteins, SCRIB24,31 and PRICKLE (Figure

2).

WNTs signaling through RTK receptors ROR and RYK

There are multiple different nodes of crosstalk between WNT signaling and

tyrosine kinases. The capacity of WNTs to directly bind and signal via

transmembrane proteins like ROR1/2, RYK and PTK7,20,32-35 which are classified

as members of the RTK family based on sequence similarity, provides one of the

most concrete examples of such crosstalk.

WNT/ROR pathway: A functional link between WNT signaling and ROR

receptors was first established in the context of normal vertebrate development. In

frogs and fish, loss of WNT5A, WNT11, and ROR2 alone or in combination leads

to defects in convergence extension, a developmental process necessary for the

elongation of the embryonic axis linked to PCP signaling. Wnt5a and ROR2

knockout mice also exhibit strikingly similar developmental defects including

cranial defects, aberrant bone formation, disrupted limb development.36,37

Numerous studies in cultured cells suggest that binding of WNTs to

ROR1/2 receptors results in the activation of numerous downstream effectors

including JNK kinases, which can phosphorylate and activate AP-1 transcription

factors in a variety of cell lines.32,33,35,38 Other studies suggest that activating

7
WNT/ROR signaling can also induce a variety of other downstream signaling

events such as the phosphorylation of DVL,35,39 the activation of PKC,40 and the

inhibition of β-catenin/TCF-dependent transcription.32,41 In some cases, ROR2

signaling involves functional interactions with FZD family receptors as the

extracellular cysteine rich domains (CRD) of FZD2 and FZD5 have been shown

to interact with ROR2 in HEK293T cells,32 while ROR2 has similarly been shown

to interact with FZD7 in mouse fibroblasts.35 Several studies also suggest

crosstalk between non-receptor tyrosine kinases and the WNT5A/ROR2 pathway.

ROR2 overexpression in mouse melanoma cells induces the phosphorylation of

both SRC and FAK,42 while treating chondrocytes with WNT5A similarly

promotes the phosphorylation and activation of SRC, which is mediated by the

formation of a ROR2-SRC complex (Figure 3A).43 .

In addition to playing critical roles in normal developmental processes,

ROR1 and ROR2 also regulate a variety of cancer cell behaviors suggesting that

increased expression of RORs observed in aggressive cancers may have

functional relevance. Inhibiting ROR1 expression with siRNAs reduces the

growth of gastric, lung, and breast cancer cells both in cell culture and in

xenografts,44-46 while knocking down ROR2 with shRNAs significantly reduces

the growth of leiomyosarcoma and renal cell carcinoma xenograft tumors in

mice.47-49 Many studies indicate an important role for ROR1 in cancer cell

survival, since ROR1 siRNAs induced apoptosis in CLL, breast, and cervical

8
carcinoma cells.45,50,51 Consistent with the described role of WNT/ROR pathway

in mediating cell migration during animal development, WNT/ROR signaling also

regulates metastatic cell behavior. Specifically, reduction in ROR2 in mouse

melanoma cells reduces the frequency and severity of lung metastases in mice,40,42

while depletion of ROR2 similarly inhibits WNT5A-induced invasion of sarcoma

cells in vitro.38,48 In contrast, ROR2 overexpression enhances leiomyosarcoma,

osteosarcoma, and renal cell carcinoma cell migration.38,48,49 Although the

mechanisms are not entirely clear, several studies suggest that ROR2 can enhance

migration by promoting the expression of matrix metalloproteases (MMPs),

which help to promote cancer cell invasion by digesting extracellular matrix

proteins.38,47-49

Importantly, numerous studies have linked ROR1/2 expression to clinical

outcomes in cancer where higher levels of ROR1/2 mRNA and protein generally

predict adverse outcomes in the clinic. ROR2 mRNA is increased in colorectal

and cervical cancer specimens compared to adjacent normal tissues,52,53 and

increased ROR2 staining in patient samples is associated with reduced overall

survival and recurrence free survival in these cancers.52,53 Other studies find that

ROR1 expression is increased in high grade and triple negative breast cancer and

high levels of both ROR1 and ROR2 predict decreased breast cancer patient

survival.45,54 Finally, ROR2 is also highly expressed in a subset of soft tissue

sarcomas where increased expression of ROR2 predicts patient deaths.48 Taken

9
together, these studies suggest that the WNT/ROR pathway might serve as a

promising therapeutic target in tumors where heightened activity is associated

with adverse clinical outcomes.

WNT/RYK Pathway: WNTs can also signal via the single pass

transmembrane receptor RYK (Receptor Tyrosine Like Receptor), which is an

unusual member of the RTK protein family due to an absence of receptor tyrosine

activity despite sequence similarity to other RTKs. Binding of WNTs to RYK

receptors is mediated by the extracellular WIF domain of RYK, which is closely

related in sequence to WIF1 (WNT inhibitory factor) a secreted protein that can

bind WNTs and inhibit their activity at the cell surface. RYK can activate both β-

catenin -dependent and independent signaling pathways.13,55-59 Although the RYK

receptor lacks intrinsic receptor tyrosine kinase activity, there is evidence that

WNT/RYK receptor complexes can mediate biological activity auxiliary proteins

such as DVL13 and SRC.60 Like ROR1/2, RYK also physically and genetically

interacts with members of the FZD family of WNT receptors,13,61 as well as other

transmembrane proteins involved in PCP signaling like VANGL2 and

CELSR1/2.56,57 Finally, RYK has been shown to undergo extensive post-

translational processing including cleavage by γ-secretase enzymes resulting in

the release and nuclear translocation of the intracellular domain of RYK,57,62 as

well as ubiquitination by the E3 ubiquitin ligase, MIB1 (Figure 3B).57

10
 The biological functions of RYK have been most extensively studied in

animal models of development. RYK has been frequently linked to the regulation

of cell polarity and migration where it often acts in a β-catenin-independent

manner. RYK genetically interacts with both WNT5 and WNT11 to regulate

convergence extension movements in developing frogs and fish.13,21 A

WNT5A/RYK pathway has also been shown to regulate axon outgrowth and

orientation in both mouse and zebra fish models of neurodevelopment.55,60,63,64 In

other contexts, RYK regulates cell migration and polarity via β-catenin-dependent

phenotypes. In cultured cells, RYK is required for WNT3A-dependent activation

of β-catenin-dependent transcriptional reporter.57,59 RYK also regulates

developmental phenotypes in a manner that involves the activation of β-

catenin/TCF-dependent transcription. In mice, RYK promotes WNT3A-induced

neurite outgrowth as well as the differentiation of neural progenitor cells,59,62

while studies in nematode worms reveal that LIN18 (a RYK homologue)

genetically interacts with WNT/MOM-2 and TCF/POP-1 to determine vulval cell

fate.58

Numerous studies also link WNT/RYK signaling to a variety of

phenotypes in cultured human cancer cells. In glioblastoma, WNT5A promoted

MMP2 expression and activity, and enhanced cell invasion through matrigel in a

manner that required RYK, but not ROR2.65 In melanoma, loss of either WNT5A

or RYK resulted in decreased cell proliferation and increased sensitivity to

11
BRAF/MAPK/ERK pathway.66 In breast cancer cells, overexpression WNT5A

suppresses cell proliferation by activating SMAD2 in a RYK and TGFβ-

dependent manner.67 Whether β-catenin -dependent or β-catenin-independent

WNT/RYK signaling can regulate cancer cell growth and metastasis in vivo

remains to be explored. Importantly, studies of possible links between RYK

receptor expression in patient samples and clinical outcomes are currently

lacking, which may be due, at least in part, to the lack of specific RYK

antibodies.57

Crosstalk between WNTs and growth factor responsive RTKs

As a second example of crosstalk between WNT and tyrosine kinase pathways,

WNT signaling pathways are also modulated by other RTKs that are not known to

directly bind WNTs, which is in contrast to ROR1/2 and RYK receptors. Many

studies suggest crosstalk between WNT signaling and signaling pathways

mediated by RTKs that bind and respond to secreted growth factors, which results

in receptor dimerization and autophosphorylation and the subsequent activation of

downstream kinases, small GTPases, and other effectors.

The hyperactivation of RTK pathways commonly observed in a variety of

different cancers may lead to increased activity of WNT/ β-catenin signaling. Co-

stimulating HEK293T cells with a variety of different RTK-stimulating growth

factors, including EGF, FGF2, and NGF, results in synergistic activation of β-

12
catenin -responsive transcriptional reporter in response to WNT3A treatment.68

HGF stimulation of both colorectal carcinoma cells and primary hepatocytes

similarly resulted in nuclear accumulation of β-catenin and increased β-catenin

target gene expression.69,70 Finally, EGF stimulation also induced nuclear

accumulation of β-catenin in neural progenitor cells.71 Activation of WNT/ β-

catenin signaling has also been observed in the presence of RTKs harboring

mutations that render then constitutively active even in the absence of growth

factor stimulation such as RON (M1254T and D1232V) and MET (M1268T)

receptors, whereas no activation of WNT/ β-catenin signaling was observed when

wild type versions of the receptors were expressed.72

Although growth factor responsive RTKs are not known to directly bind

WNTs, they can still enhance WNT signaling through indirect mechanisms such

as enhancing the activity of downstream kinase pathways. Depending on the

particular cell type, growth factor/RTK signaling seems to enhance WNT

signaling through different pathways. Specifically, FGF2 mediated-enhancement

of WNT3A/ β-catenin -dependent transcription in HEK293T cells was not

affected by inhibitors of PI3K, but could be blocked by treatment with small

molecules to inhibit MEK/ERK signaling.68 In contrast, the stimulation of β-

catenin activity with HGF could be blocked by treatment with PI3K inhibitors,

but not with mTOR or MEK/ERK inhibitors.69 Some studies suggest that

activation of growth factor-responsive RTKs through ligand binding can also

13
regulate the phosphorylation status of various WNT signaling proteins. For

example, FGF2 stimulation was shown to induce ERK-dependent

phosphorylation of the WNT co-receptor LRP6 phosphorylation at Ser1490 and

Thr1572, but did not affect WNT3A-dependent phosphorylation at Ser1490.68

Enhancing FGF2/FGFR, EGF/EGFR, and NGF/TRKA also increased ERK

phosphorylation and synergized with WNT3A to activate β-catenin -dependent

transcriptional reporter,68 raising the possibility that multiple different

RTK/ligand combinations can regulate WNT3A signaling via ERK

phosphorylation of LRP6.

β-catenin is a multifunctional protein that can associate with multiple

protein complexes including the destruction complex, transcription complexes

induced upon WNT stimulation, and junctional complexes at the plasma

membrane where β-catenin interacts with proteins such as α-catenin and E-

cadherin to mediate cell-cell adhesion. Activation of RTKs in cancer can also

influence WNT/ β-catenin signaling by regulating protein complexes especially

those involving β-catenin. Specifically, expression of hyperactive mutant MET

and RON RTKs disrupted the AXIN-/GSK3β/β-catenin complex required for

proteasome-dependent degradation of β-catenin,72 while IGF-II treatment induces

rapid loss of E-cadherin at junctions and results in the accumulation of both β-

catenin and TCF3 in the nuclei in rat bladder carcinoma cells.73 In C10 colorectal

cells that lack β-catenin and APC mutations, IGF-1 stimulation similarly resulted

14
in decreased association between E-cadherin and β-catenin.74 The association

between β-catenin and E-cadherin in C10 colorectal cells could also be modulated

inhibitors of tyrosine phosphatases but not with serine phosphatase inhibitors,74

suggesting that the interaction between β-catenin and E-cadherin is dependent on

tyrosine phosphorylation. However, stimulation of RTKS with growth factors

does mediate the association between β-catenin and E-cadherin in all contexts as

HGF stimulation of hepatocytes increased nuclear β-catenin, but had no effect on

E-cadherin/ β-catenin interaction.70

WNT signaling and non-receptor RTKs

In addition to either direct or indirect interactions with receptor tyrosine kinase

family members, numerous studies point to functional crosstalk between WNT

signaling and phosphorylation networks regulated by non-receptor tyrosine

kinases, a diverse protein family which includes 32 different cytosolic enzymes.

Non-receptor tyrosine kinases have been shown to mediate the effects of both β-

catenin -dependent and –independent WNT signaling pathways.

For example, several studies using cultured cancer cells suggest that SRC

family kinases can enhance WNT/ β-catenin signaling. Overexpression of SRC

enhanced, whereas siRNA-mediated depletion of SRC reduced WNT3A-induced

β-catenin -dependent transcriptional reporter activity in embryonic carcinoma

cells.75 Importantly, the inhibition of β-catenin-dependent transcription

15
overserved following SRC depletion could be rescued by co-expression of wild

type SRC, but not kinase dead SRC suggesting that SRC kinase activity plays an

important role in mediating WNT3A responsiveness.75 Moreover, co-transfection

of HEK293T cells with a S37A β-catenin mutant , which is constitutively

stabilized and does not require WNT for activation with vSRC resulted in

increased β-catenin -dependent transcription,76 indicating that SRC can function

downstream of the β-catenin destruction complex. In further support of a role for

SRC downstream of the destruction complex, vSRC could also activate β-catenin

-dependent transcription in SW480 colorectal cells, which have a stabilized β-

catenin due to a mutation in APC preventing normal function of the destruction

complex.76 This study went on to show that the enhancement of β-catenin -

dependent transcription by vSRC could be inhibited by co-transfection with a

dominant negative TCF4 that can prevent TCF4-dependent transactivation of β-

catenin target genes.76

One way that SRC family kinases can regulate β-catenin signaling is

through the direct phosphorylation of β-catenin. In Vitro studies using purified

proteins reveal that cSRC modifies Tyr-86 and Tyr-654, located in the N-terminal

domain of β-catenin.77 Other SRC family kinases such as FYN, YES, and FER

have also been shown to phosphorylate β-catenin in vitro.78 As opposed to

phosphorylation of β-catenin by GSK3beta, which targets it for degradation,

phosphorylation of β-catenin by SRC family kinases seems to enhance the

16
transcriptional activity of β-catenin. Specifically, overexpression of a Y654E

phosphomimetic version of β-catenin caused a greater increase in β-catenin -

driven transcriptional reporter activity than compared to overexpression of wild

type β-catenin.77 In vitro, SRC-mediated phosphorylation of β-catenin enhances

binding to TATA binding protein (TBP), a general activator of RNA polymerase

II transcription.77 Taken together, these studies reveal that SRC promotes WNT/

β-catenin signaling at the level of β-catenin /TCF4 transcription complexes.

In addition to regulating the composition of β-catenin transcriptional

complexes, Tyrosine phosphorylation of β-catenin may also regulate the

subcellular localization of β-catenin in a manner similar to RTK activation.

Several studies suggest that tyrosine phosphorylation of β-catenin reduces the

association with junctional protein complexes. Overexpression of FER or FYN

decreased the association between β-catenin and alpha-catenin in RWP1

embryonic retina cells.78 Activation of receptor tyrosine signaling by

overexpression of RET oncoproteins also caused a dissociation between β-

catenin and E-cadherin and resulted in an increase in β-catenin association with

p300,79 a lysine-specific acetyltransferase that acts as a positive regulator of β-

catenin -dependent transcription. Importantly, not all cell types have both

junctional and cytosolic/nuclear pools of β-catenin, so this mechanism of

crosstalk between SRC family kinases and β-catenin signaling may be relevant

17
only in the cells where junctional β-catenin complexes are formed, which are

usually epithelial in nature.

In some contexts, crosstalk between the WNT/ β-catenin signaling

pathway and SRC may also involve SRC-dependent phosphorylation of other

WNT signaling proteins such as DVL and LRP6. SRC and the SRC family

kinases interact with DVL2 via the SH3/SH2 domains of SRC in GST-pulldown

experiments using purified proteins.75 SRC could also interact with DVL2 in

cultured cells in co-immunoprecipitation experiments, and this interaction

between DVL2 and SRC as well as the phosphorylation of SRC were induced

upon stimulation with WNT3A.75 SRC phosphorylates DVL2 at multiple tyrosine

residues in vitro75 and while overexpressing wild type DVL2 enhanced WNT3A

induced TOPFLASH; this enhancement was reduced when tyrosines targeted by

SRC were mutated (Y27F, Y18F, Y275F),75 suggesting that DVL

phosphorylation is required for SRC-dependent enhancement of WNT signaling.

Interestingly, in other cell types, SRC has been shown to inhibit WNT/ β-

catenin signaling, potentially through the modulation of LRP6 phosphorylation.

Specifically, both SRC and FER have also been shown to phosphorylate LRP6 in

vitro and overexpression of wild type SRC, but not kinase dead SRC inhibits the

activation of β-catenin -dependent transcriptional reporters induced by the

overexpression of LRP6 in HEK293T cells.80 In developing zebrafish embryos

overexpression of LRP6 results in defects in anterior-posterior patterning, and co-

18
injection of LRP6 mRNA with SRC mRNA partially reversed the LRP6-

dependent patterning phenotype indicating that SRC can also inhibit LRP6

function in vivo.80

Other non-receptor type tyrosine kinases can similarly interact with the

WNT/β-catenin signaling pathway. The non-receptor tyrosine kinase PYK2 is

overexpressed in multiple myeloma samples measured at both a protein and RNA

level was recently shown to act as a positive regulator of β-catenin -dependent

transcription.81 Analysis of RNA expression revealed that knockdown of PYK2

also reduced the abundance of known β-catenin transcriptional targets, while

overexpression of PYK2 had the opposite effect.81 Mechanistically, depletion of

PYK2 resulted in a loss of β-catenin stability via increased activity of the

destruction complex.81 Knockdown of PYK2 also resulted in increased survival

and decreased tumor burden in a murine xenografts model of multiple myeloma,81

suggesting that PYK2 has an important role in myeloma carcinogenesis.

Studies using vertebrate models of developmental model systems also

suggest that WNT5A and WNT11, two WNTs that generally signal through β-

catenin -independent signaling networks, require the functions of SRC family

tyrosine kinases for their activity. Knockdown of the non-receptor tyrosine

kinases FYN and YES in zebrafish using morpholinos results in convergence

extension defects resulting in reduced extension of the embryonic axis, which

phenocopy developmental defects observed following knockdown of WNT11.9

19
Importantly, combining a low dose of WNT11 with FYN/YES morpholinos that

did not result in phenotypic abnormalities on their own resulted in convergence

extension phenotypes, suggesting a genetic interaction between WNT11 signaling

and FYN/YES.11 However, the mechanisms by which FYN/YES can promote

WNT5A and WNT11 induction of β-catenin -independent signaling are not yet

clearly defined. It is possible that the ability of SRC family kinases to regulate

both β-catenin–dependent and –independent WNT signaling networks may

depend on the levels and activity of WNT signaling proteins present in a

particular cell or tumor type.

Targeting crosstalk between WNTs and tyrosine kinase signaling as a

therapeutic strategy

Although our understanding of both the mechanisms and the functional

consequences of crosstalk between WNT signaling and tyrosine kinase signaling

remains incomplete, there are already a number of studies suggesting that

crosstalk between these pathways may be relevant in the clinic. Tyrosine kinases

have been successfully targeted in the clinic using a variety of different strategies

including small molecules as well as blocking antibodies for RTKs. Although

WNT signaling pathways have been difficult to modulate pharmacologically in

the past, a number of strategies for modulating WNT signaling that similarly

20
range from small molecules, to blocking antibodies, to peptide-based inhibitors

have recently emerged.

As predicted by numerous studies suggesting signaling crosstalk between

WNTs and tyrosine kinases, treating cancer cells with various inhibitors of

tyrosine kinase signaling can result in alterations in WNT signaling activity. For

example, although sorafenib resulted in reduced β-catenin-dependent

transcriptional activity in xenograft model of hepatocellular carcinoma,82 however

it was not clear whether decreased β-catenin signaling was a pre-requisite for

sorafenib response. Treatment of myeloma cells with a PYK2 inhibitor VS-4718

(Verastem) similarly resulted in decreased β-catenin protein abundance and target

gene expression, which correlated with decreased disease progression in a mouse

xenograft model of myeloma.81 Finally the Bruton’s Tyrosine Kinase (BTK)

inhibitor CGI1746 inhibits the growth of myeloma cells grown both in vitro and

in a mouse model of myeloma and can also regulate the activity of β-catenin.83

However, it is also not yet clear whether crosstalk with WNT/ β-catenin signaling

is necessary for the therapeutic efficacy of BTK inhibitors. Moreover, loss of

BTK function in colorectal cancer cells, B cells and zebrafish embryos resulted in

elevated WNT/ β-catenin signaling, suggesting that BTK acts as a negative

regulator of the pathway in some contexts.84

21
 Some studies also suggest that co-targeting both WNT and tyrosine

kinases may result in better outcomes than targeting one pathway. For example, a

genome-wide shRNA screen for regulators of gefitinib responsiveness in NSCLC

cell lines identified multiple genes linked to WNT signaling: TCF3, DVL3,

SMAD3, CCND1, WNT2B, PP2A, and DVL3, as well as FZD3.85 Expression

profiling of HNSCC after cMET inhibition with PF-2341066 identified the

WNT/β-catenin cascade as the most downregulated canonical pathway.86

Inhibiting WNT/β-catenin signaling by overexpressing negative regulators of the

pathway (DKK1 and AXIN) inhibited spheroid formation by HNSCC in both the

presence and absence of HGF to stimulate cMET and other RTKs,86 suggesting

that inhibition of β-catenin signaling may be a good strategy for HNSCC with

activated cMET signaling. β-catenin also interacts with the with Bcr-Abl fusion

protein, a key driver of CML tumorigenesis.87 This formation of β-catenin and

Bcr-Abl protein complexes could be inhibited by treatment with the Bcr-abl

inhibitor imatinib, or the SRC family kinase inhibitor SU6656, suggesting that

Bcr-Abl phosphorylation is necessary for interaction with β-catenin.87 Crosstalk

between β-catenin and Bcr-Abl has important functional complexes in CML since

β-catenin and Bcr-Abl appear to cooperate to promote CML cell survival and

growth. Specifically, combining imatinib and either a dominant negative TCF4 or

β-catenin siRNAs to inhibit β-catenin -dependent transcription caused a greater

inhibition of proliferation and an increased induction of apoptosis compared to

22
either imatinib treatment or inhibiting β-catenin alone.87 Finally, other studies

raise the possibility that the expression of WNT signaling proteins could be used

as biomarkers to better predict patient responses to therapies targeting tyrosine

kinases. For example, DVL3 was identified as one of the factors mediating tumor

cell responsiveness to the IGFIR tyrosine kinase inhibitor (TKI) AZ12253801 in

prostate and breast cancer cells.88 Importantly, DVL3 staining in patient tumors

was found to predict patient drug responses to IGFIR-targeted therapy.88

Final Thoughts

Aberrant hypo- or hyperactivity of signal transduction pathways is a hallmark of

cancer. Multiple mechanisms drive cancer-specific alterations in signal

transduction including genetic mutations in signaling molecules, aberrant control

of gene regulatory networks, and changes in the stability, localization, and

covalent modification of signaling proteins. In the clinic, signal transduction

pathways have been targeted by small molecules, targeting antibodies, and other

interventions sometimes leading to robust responses. Despite these successes,

monotherapies aimed at a single molecule or pathway are not universally

effective, and are almost always combined with other interventions such as

generic DNA damaging agents and radiation therapy, which cannot be tolerated

by all patients. As we enter a new era of personalized medicine, it is now clear

that each cancer patient is unique and that multiple different pathways are often

23
simultaneously up- or down-regulated in tumor cells. Furthering our

understanding of the mechanisms of crosstalk between WNT signaling, tyrosine

kinase signaling, and many other different pathways remains a key challenge that

must be surmounted in our efforts to understand the mechanisms of cancer

initiation and progression and to apply this knowledge to the development of

patient-specific therapeutic strategies.

24
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Figure 1: β-catenin-dependent WNT signaling

A) In the absence of WNT stimulation the abundance of the transcription factor

β-catenin (CTNNB1) is kept low by the activity of the destruction complex,

which is made up of the scaffolding proteins AXIN1/2 and APC, the kinases

GSK3β and CKII, and the ubiquitin ligase TRCP among other factors. This

destruction complex promotes the phosphorylation, and subsequent

ubiquitination and degradation of β-catenin by the proteasome. In this “off”

state the TCF/LEF family of transcription factors acts to repress gene

transcription in cooperation with other repressive proteins like histone

deacetylases (HDACs). B) WNT/ β-catenin signaling is activated when

WNTs bind to a co-receptor complex comprised of FZD and LRP5/6, which

leads to the activation of DVL, and the inhibition of the destruction complex,

which is recruited to the plasma membrane. Consequentially, β-catenin

becomes stabilized and translocates to the nucleus where it can cooperate

with members of the TCF/LEF family of transcription factors resulting in the

recruitment of a transcriptional co-activator complex and the subsequent

activation of a variety of target genes involved in orchestrating different cell

phenotypes such as proliferation, differentiation, and migration.

41
Figure 1: β-catenin-dependent WNT signaling

A) In the absence of WNT stimulation the abundance of the transcription factor

β-catenin (CTNNB1) is kept low by the activity of the destruction complex,

which is made up of the scaffolding proteins AXIN1/2 and APC, the kinases

GSK3β and CKII, and the ubiquitin ligase TRCP among other factors. This

destruction complex promotes the phosphorylation, and subsequent

ubiquitination and degradation of β-catenin by the proteasome. In this “off”

state the TCF/LEF family of transcription factors acts to repress gene

transcription in cooperation with other repressive proteins like histone

deacetylases (HDACs). B) WNT/ β-catenin signaling is activated when

WNTs bind to a co-receptor complex comprised of FZD and LRP5/6, which

leads to the activation of DVL, and the inhibition of the destruction complex,

which is recruited to the plasma membrane. Consequentially, β-catenin

becomes stabilized and translocates to the nucleus where it can cooperate

with members of the TCF/LEF family of transcription factors resulting in the

recruitment of a transcriptional co-activator complex and the subsequent

activation of a variety of target genes involved in orchestrating different cell

phenotypes such as proliferation, differentiation, and migration.

42
Figure 2: β-catenin-independent WNT signaling pathways

Some WNTs act independently of CTNNB1 transcriptional activity in order to

regulate cell behaviors such as the establishment of planar cell polarity (PCP), or the

orientation of cells within a tissue plane. The PCP signaling network shares some

common features with β-catenin -dependent signaling including the formation of

WNT/FZD receptor complexes and a requirement for DVL function. However, these

β-catenin-independent pathways are also unique given that the function of LRP5/6

co-receptors and the proteins that make up the destruction complex are not required.

WNT/PCP signaling is mediated by additional factors including transmembrane

proteins like VANGL1/2, CELSR1/2, PTK7, and cytosolic factors such as SCRIB

and PRICKLE that are not required for β-catenin -dependent signaling. In addition to

signaling through the PCP pathway, β-catenin-independent WNTs can also regulate

the activity of various kinase cascades such as the JNK pathway resulting in the

activation of AP-1 transcription factors, as well as Ca2+-responsive signaling

networks. Finally, activation of both the PCP pathway and other β-catenin-

independent WNT can lead to changes in the activity or subcellular localization of

small GTPases such as RHOA, RAC1, and CDC42, which play critical roles in the

regulation of cell invasion and metastasis in cancer.

43
44
45