WNT

DISCOVERY
1. Integration of mouse mammary tumour virus (MMTV) into the promoter of a gene called Int-1-induced tumours.
2. Int-1 is orthologous to the Drosophila segment polarity gene Wingless (Wg)
3. The terms were combined to produce the name Wnt.
4. The WNT gene family currently comprises 19 paralogous members.
FUNCTIONS
1. Wnt signalling plays key role during embryogenesis
2. Embryonic patterning through the control of cell proliferation and determination of stem cell fate.
3. In mature tissues, Wnt signalling is essential for the maintenance of normal architecture and function of many tissues
through the control of stem cell renewal.
4. In animals, Germ-line alterations of Wnt signalling lead to a variety of morphological abnormalities.
5. In humans, germ-line mutation of Wnt signalling genes leads to congenital defects.
6. In mature tissues, activation of Wnt signalling via somatic mutations lead to cancer.
Morphogens Porcupine
• Wnt, are activated by morphogens govern the
dev. of tissues.  Porcupine is Required to Secrete Wingless.
• forms a concentration gradient across dev. tissues.  Encodes multipass TM ER prt for wg dev in
• signalling molecules act directly on cells to embryo
produce cellular response.  No Porcupine-no wg secretion
•

Epistasis Components:
• Epistasis: the loss of one gene masks the effect of
the loss of another • Wnts
• Double mutant-when 1 mutant masks the • Extracllular Antagonists: Frzbs, Dikkopf
phenotype of the other. • Receptors: Frizzled, Arrow,LRP5/6
• • Cytoplasmic: Dsh, APC, Axin, ZW3, Arm
• Nuclear: Groucho, TCF, Arm, Pan, Lgl

WNT PROTEINS
1. The Wnt proteins are a family of small (39–46 kDa) lipid modified secreted glycoproteins
(Made of 350-400 a.as).
2. They contain 23–24 cysteine residues with highly conserved spacing
3. Wnt proteins are ligands for the Frizzled family of seven-pass transmembrane receptors.
4. Wnt proteins contain an extracellular cysteine-rich domain (CRD) and an intra cytoplasmic
PDZ domain binding motif.

Frizzleds and FRP/FrzBs

• 7 Transmembrane domains
• Conserved Cysteine Rich Domain (CRD), binds Wnts
• At least 11 vertebrate Fzs
• Secreted FRP/FrzB proteins modulate signalling
• Extracellular loops may also bind Wnt and activate signalling
Dickkopf and Kremen Function

Arrow/LRP5/6
• Arrow is a fly mutant, LRP5/6 refers to the vertebrate homologues: Low density lipoprotein receptor Related Protein
(LRP)
• Single transmembrane protein, binds to Wnt and forms tertiary complexes with Fz and Wnt
• An extracellular repressor of signalling, Dickkopf, binds Arr/LRP

Dishevelled
• Cytoplasmic protein, three conserved domains: DIX, PDZ and DEP
• 18 possible protein interactions identified
• Binds to FRAT/GBP which binds to GSK3
• Phosphorylated by several kinases
• Associated with the membrane on signalling, interacts with Fz
Axin APC
• Product of the mouse fused locus • Adenomatous Polyposis Coli
• Antagonises Wnt signalling • Major tumour suppressor
• Regulator of G-Protein Signalling (RGS) domain • Multiple cellular roles: also regulates
and DIX domain microtubules
• Huge protein, many domains and protein
interactions
• Antagonises Wnt Signalling

Zest-white3/Shaggy/GSK3 Armadillo/-Catenin
• Glycogen Synthase Kinase 3 (GSK3) • Roles in cell adhesion as part of the
• A sereine/threonine kinase cadherin/catenin junctional complexes
• Long known to phosphorylate Armadillo, now • Key mediator of Wnt/Wg signalling
also known to phosphorylate APC, Axin and Arr • In absence of signalling cytoplasmic levels are
• Antagonises Wnt signalling low, during signalling levels are increased
• • Activates transcription but can’t bind DNA

TCF and Groucho Minimal Physical Interactions

• T-cell factor, can bind DNA but can’t activate • Wnt: Fz and Arr
transcription • Fz: Dsh
• In the absence of signalling they repress • Arr: Axin
transcription of target genes in concert with • Dsh: Axin and GBP
groucho • Axin: APC: -catenin
• In the presence of signalling b-catenin displaces • GSK3: GBP, Axin and -catenin
Groucho from TCF and turns on transcription at • -catenin: TCF
the same promotors The Destruction Complex
• When Wnt signalling is inactive, APC and Axin
Pygopus and Legless/Bcl9 act as scaffolding proteins to bring GSK3 and
• Legless/Bcl9 links b-catenin to Pygopus which is other kinases into proximity with -catenin
required for the transcription of Wnt responsive • Phosphorylation of -catenin tags it for
genes destruction by the proteosome
WNT PATHWAY
On receptor–ligand interaction, one of three different signalling pathways can be activated.

1. The canonical Wnt signalling pathway that results in stabilization and increased transcriptional activity of β- catenin.
This Wnt signalling pathway is involved in cancer development.

2. The Wnt/ planar cell polarity (Wnt/ PCP) pathway mediated through activation of the c-Jun N-terminal kinase (Jnk)
pathway and is considered to regulate the cytoskeleton and cell polarity.

3. The Wnt/calcium (Wnt/Ca2+) pathway that is activated through heterotrimeric G proteins and results in increased
intracellular calcium and activation of protein kinase C (PKC) but of unknown function.

Each pathway appears to be transduced initially through the cytoplasmic protein Dishevelled. The availability of ligands and
the pattern of expression of cell surface receptors is probably the most important factor of cellular response during Wnt
signalling.The uncanonical pathway is uncertain as it can antagonize or activate the canonical pathway, and can independently
promote tumour progression.

THE CANONICAL WNT SIGNALLING PATHWAYS

In the presence of wnt,

1. Canonical Wnt signallings is only possible through formation of a trimeric complex consisting of
 Wnt ligand,
 Frizzled receptor,
 Cell surface receptor low-density lipoprotein receptor–related protein (LRP)5/6.
2. Canonical signaling is initiated when Wg/Wnt ligands binds to Frizzled (Fzd)/ low density lipoprotein receptor related
protein LRP receptors.
3. This activates the cytoplasmic protein dishevelled (Dsh in Drosophila and Dvl in vertebrates) partly due
Phosphorylation by casein kinase 1 (CK1) and casein kinase 2 (CK2).
4. Activated Dsh/Dvl then inhibits the activity of the multiprotein complex
 b -catenin
 Axin
 adenomatous polyposis coli (APC)
 glycogen synthase kinase (GSK)-3 b) (GBP)
which targets b -catenin by phosphorylation for degradation by the proteasome.

5. This results in accumulation of cytosolic b-catenin.

6. β- Catenin protein is normally found at low levels in the cytoplasm but activation of Wnt signalling will raise β-
catenin levels.
7. The β-catenin, stabilized under Phosphorylation will then translocate into the nucleus and bind to members of the T-
cell factor (Tcf)/Lymphoid enhancing factor (Lef) family of DNA binding proteins leading to transcription of Wnt
target genes.
8. Thus ligand binding triggers a series of intracellular events that lead to inhibition of the cytoplasmic β - catenin
destruction complex.
9. Pygo and Lgs/BCL9 are recently identified nuclear co-factors of b-catenin
10. This activity causes tumour. Thus it is essential that Wnt signalling is tightly controlled.
11. Regulation of Wnt signalling occurs at several different levels to ensure that cytoplasmic levels of free β-catenin
protein remain low.

In the absence of wnt,

1. β-catenin is attached to the membrane by E-cadherin and α-actin.

2. A complex of Axin, APC and the serine-threonine kinase GSK3b binds and phosphorylates cytoplasmic b-catenin.
3. This triggers ubiquitination by an E3 ligase complex containing b-TrCP, resulting in proteolytic degradation of b-
catenin.
4. Phosphorylated b-catenin is recognized by b-transducin repeat-containing protein (b-TrCP) and degraded by the
proteaosome.
5. Studies have shown that if any one of the four proteins in the degradation complex is mutated, the intracellular
concentration of β-catenin can lead to a form of cancer.
At the Cell Surface
Activation of Wnt Signalling (the ‘‘On’’ Switches)
Wnt ligand binds with frizzled receptors in the
presence of LPR5/6 transmembrane protein. (Tri
molecular complex is formed wnt-friz-LRP5/6). 3
steps:
1. Dishevelled is recruited to the cell surface from
cytoplasm and phosphorylated by casein kinase
1 CK1.P-DSV binds to FRAT 1 binds and
inhibits GSK beta.
2. wnt-friz-LRP5/6 results in p-of LRP5/6 by
GSK beta and CK1.Doubly p- LRP5/6 disrupts
GSK beta/axin complex and recruits axin to
cell surface and degrades it. This result in
destabilization and increase in β-catenin levels
as β-catenin levels is not p-. β-catenin is not
ubiquitinated and thereby escapes proteasomal
degradation. And its translocation to the
nucleus.
3. Increase in β-catenin has gr8ter affinity to
TCFS than cadherin. C-terminus of β-catenin
binds to TCF and leads to conformational
change. C-terminus of β-catenin flips causing
steric hindrance for cadherin. Thus β-catenin
binds with cadherin in the cytoplasm leaving
TCF unaffected. Proteins pygopus and
Bcl9/legless forms a complex with β-catenin in
the cytoplasm Then β-catenin is released
(free).Many RTK binds with their ligands and
p-tyrosine residue serine 120 of β-catenin. Thus
dissociation of β-catenin from the cadherin–
catenin complex is seen. And recycled to the
cytoplasmic pool, which is then followed by
increased expression of β-catenin target genes.
Inhibition of Wnt Signalling (the ‘‘Off’’ Switches)
Wnt antagonists
Mechanisms to inhibit Wnt signalling at the cell
surface .These include
1. reducing the amount of Wnt ligand,
2. reducing the level/activity of
receptors, and
3. sequestering β-catenin from the
cytoplasmic pools in multiprotein
complexes within the cell
membrane.
1. secreted frizzled-related proteins (sFRPs)
(act as Wnt agonists) binds with Wnt ligand
and prevent it from binding with its receptor.
2. Wnt-inhibitory factor-1 (WIF1)contains
conserved WIF domain with five epidermal
growth factor (EGF)-like repeats.
3. Dickkopf (Dkk) Reduces the cell surface
LRP5/6 receptor by acting as antagonize
Wnt signalling through inactivation of the
surface receptors LRP5/6 .The Dkks form a
ternary complex with LRP5/6 and the single
pass transmembrane receptors Kremen that
undergoes endocytosis, thereby removing
LRP5/6 receptors.
4. β-catenin forms complex with E-cadherin
and α-catenin in cell surface and control cell
adhesion.Thus Cadherins can inhibit Wnt
signalling.
Increase in surface E-cadherin expression will deplete
cytoplasmic free β-catenin and thereby inhibit Wnt
signalling which represents a feedback mechanism to
control levels of cytoplasmic β-catenin.
In the Cytoplasm
Activation of Wnt Signalling (the ‘‘On’’ Switches)
β- Catenins are stabilized and translocated to the nucleus due to
p-of various residues in β- Catenin. Thus attains the
susceptibility for degradation. Various residues that p- β-
Catenin
1. PKA :p- β- Catenin at serine 552 and 675.Thus
leads to stabilization of β- Catenin and inhibit
ubiquitination.
2. CK2:p- β- Catenin at threonine 393.Thus prevents
axin mediated degradation.
 PK, IĸBkinaseα: inhibits Axin/APC/GSKβ and siah1
pathway of β- Catenin degradation
 Kinase independent stabilization also occurs.
 E2, Prostaglandin(metabolite
cyclooxygenes2):interacts with their receptors and
stimulate wnt signalling.
 Receptor ligand interaction causes
1. associates of g-protein α unit
with Axin
2. dissociation of β- Catenin
destruction complex
 various transport protein translocate β- Catenin from
cytoplasm to nucleus as β- Catenin (both terminus)
has nuclear localization activity/signal NLA/NLS.
-APC and
-TCF-4(transcriptional cofactor).BCL-9/Legless
compete with β- Catenin and
-Pyrogus
translocate β- Catenin from cytoplasm to nucleus.
Inhibition of Wnt Signalling (the ‘‘Off’’ Switches)
Phosphorylation-dependent β-Catenin Degradation
Axin/APC/GSK3β mediated destruction is the main
mechanism for controlling levels of β-catenin
1. β- Catenin is constitutively produced and has a half-
life of less than 60 minutes.
2. Axin forms a complex with β-
Catenin/APC/GSKβ/CK1/PP2A protein phosphatase
3. APC has binding sites for β-Catenin and PP2A while
GSKβ/ has binding site for Axin.
4. Complex formed is stabilized by GSKβ mediated p-
of Axin and APC and PP2A with B56 subunit.
5. Within the complex, GSKβ p- N-terminus of β-
of Catenin (has regonition site for GSKβ b/w a.as 33-
45 containing 4 serine and threonine residues)
6. Serine 45 p-by CK1
7. β-transducin repeat containing protein (TrCP)
-is a ubiquinated protein
-f-box containing protein with
SIP,EBI,skpl,Cullen,Rbx-1,Enz ubiquitin ligase (E3),
ubiquitin conjugated Enz (E2), ubiquitin activating Enz
(E1).
8. P- β- Catenin is regonized by TrCP due to a.as 32-
37 in β- Catenin and ubiquitinated at 19 lysine
residues and destroyed/degraded by the
proteaosome.
Phosphorylation-independent β-Catenin Degradation
Independent GSK3β-mediated Phosphorylation
1. Siah1 is induced by p53, β-catenin and the C-terminus of
APC. This reduces the levels of cytoplasmic β-catenin.
2. Siah1 is found to be associated with ubiquitin conjugated
Enz (E2), and a Enz ubiquitin ligase (E3).
3. β-transducin repeat containing protein (TrCP)
-is a ubiquinated protein
-f-box containing protein with EBI,skpl,Cullen,Rbx-
1,Enz ubiquitin ligase (E3), ubiquitin conjugated Enz (E2),
 ubiquitin activating Enz (E1).
4. EBI associates with β-catenin,
5. SIP associates with Siah1
6. Thus whole complex can cause ubiquitination of β-catenin
without GSK3β-mediated phosphorylation.
7. p53 is found to be a β-catenin target gene and this may
represent a feedback loop.

In the nucleus Inhibition of Wnt Signalling (the ‘‘Off’’

Activation of Wnt Signalling (the ‘‘On’’ Switches) Switches)
Cytoplasmic β-catenin translocate to the nucleus
1. β-Catenin competes with Groucho for binding Cytoplasmic β-catenin translocate to the
with the LEF/TCF proteins nucleus
2. The LEF/TCF proteins allow β-catenin to bind 1. Without Wnt ligand stimulation,
to the DNA. it is insufficient to activate target
3. Thus forms a large complex of molecules that gene transcription.
allows specific target genes to be transcribed. 2. β-Catenin cannot bind to DNA
They are at the consensus motif
 essential cofactors( (A/T)(A/T)CAA(A/T)G to
pygopus and Bcl-9/legless) activate transcription.
 proteins (such as 3. It complex with members of the
p300/Creb binding protein LEF/TCF family of high
(CBP) mobility group (HMG) proteins.
 Pontin52 4. This provides a DNA binding
4. Specific target genes to be transcribed by domain for β-catenin.
various mechanisms 5. The LEF/TCF family consists of
- Histone modification of chromatin four proteins
-acting as bridges between β-catenin and (LEF-1, TCF1, TCF3,
the transcriptional machinery. and TCF4)
5. β-catenin targets genes are as c-myc, c-Jun, and 6. In the absence of nuclear β-
Sox9. catenin, the LEF/TCF proteins
6. In turn,these genes alter the expression of their are found in complex with
own target genes. transcriptional repressors such as
 Groucho and CtBP .
Feedback Loops and Other Signalling Pathways 7. Histone deacetylases
Positive feedback loops that enhance the Wnt signalling (transcriptional repressors), are
pathway. also recruited to the complex,
to maintain gene repression.
1. Increased levels of β-catenin can increase Lef-1 8. Chibby and Sox proteins (
splice variant(favours further β-catenin-mediated HMGproteins), competes with
transcription ) LEF/TCF proteins for β-catenin.
2. Increased levels of CKII after Wnt stimulation 9. Duplin and ICAT ( β-catenin
can stabilize β-catenin protein. binding proteins) prevents
3. Integrin signalling, can cause nuclear formation of β-catenin/TCF
localization of β-catenin complexes.
4. Few that activates Wnt signalling by inhibiting 10. β-catenin/TCF complexes
GSK3β binding to DNA is inhibited by
insulin-like growth factor (IGF) signalling p- of TCFs by Nemo-like kinase
Kras signalling ( to facilitate stabilization of β- (Nlk).
catenin)
EBV Feedback Loops and Other Signalling
5. STAT3 signalling can stimulate nuclear Pathways
translocation of β-catenin. Negative feedback loops that inhibits the Wnt
signalling pathway.

1. Wnt signalling can be up

regulated by the expression of
inhibitory molecules (DKK-1,
Axin2, β-TrCP, E-cadherin, and
NLK).
2. Wnt signalling is modulated and
inhibited by other signalling
pathways (BMP, TGFβ, Notch,
and Hedgehog signalling
Pathways).
3. Non canonical Wnt signalling can inhibit
canonical Wnt signalling.

Wnt signaling: Oncogenes and Tumour Suppressors

Oncogenes- wnt and β-catenin (over exp of both leads to constitutive activation of the pathway)

Tumour Suppressors - APC,AXIN,TCF

Wnt SIGNALLING AND CANCER

In tumours, Wnt signalling is in a constant “On” state, resulting elevated levels of β-catenin and a constant
aberrant expression of target genes.All mutations occur in exon 3 and specifically disrupt GSK3β-mediated
phosphorylation. The effect of the mutations is to produce a protein that is transcriptionally active but which
cannot be degraded by the APC–Axin complex.

1. Mutations of the β-catenin gene (CTNNB1 in exon 3) occur frequently in many types of cancer.
-Missense mutations and small in-frame deletion. -catenin mutated in many cancers BCL1 – B-
cell Lymphoma 1
• -catenin Activation- Loss of phosphorylation sites in cancer render it refractory to destruction.

2. Mutations in Frizzled and LRP5 surface receptors, and other Wnt proteins as it is constant “On”
state
3. Molecules that inhibit Wnt signalling are inactivated through either loss-of-function mutations or
epigenetic silencing.
4. APC cloned as the gene associated with Familial Ademotosis Polyposis (FAP-colon cancer)
Inherited predisposition to colorectal cancer.80% of human colon cancers have inactivated
APC.The rest have activated -catenin
5. When APC IS lost , destruction complex can’t form, -catenin isn’t phophorylated and so
constitutively activates transcription
6. Mutations in APC and AXIN1/2 (are integral to the β-catenin phosphorylation complex) cause
destabilization of the complex.
7. Somatic APC mutations will result in loss of the Axin binding sites.This shows that loss of β-
catenin regulation is the major selective drive for APC mutations.
8. The sFRPs, that inhibits Wnt signalling, at the cell surface under goes epigenetic silencing thus
inhibited in tumours by promoter hypermethylation. Reactivation of sFRPs with demethylating
agents has an inhibitory effect on tumour cells.
9. Many cancers have CTNNB1 mutations.
10. There are some differences between tumours with regards to mechanism for activating Wnt
signalling.
80% of colorectal tumours have APC gene mutation
(APC is a tumour suppressor requiring at least two mutagenic events for complete loss of APC
activity. APC is a large multifunctional protein and mutation will lead to the loss of other functions
in addition to that of controlling β-catenin levels.)
12% contain CTNNB1 mutations
(a single mutation of CTNNB1 is required for activation of β-catenin)
11. Mutation of the β-catenin gene alone is sufficient for adenoma development in the intestine.
12. APC mutations are rarely colon.
13. Activation of Wnt signalling (through mutation of either AXIN1/2 or CTNNB1) is common in
human hepatocellular
carcinomas.
• Axin Loss- Human Axin may be a tumour suppressor like APC.Mutants identified in human
hepatocellular carcinomas (Satoh et al, 2002)
• T-Cell Factor: TCF- Originally cloned as Lymphoid transcription factors.Tcf1 mutant mice
develop adenomas in the gut and mammary glands (Roose et al 1999).Introduction of a mutant
APC allele into these mice increases the number of tumours
• Other Signalling Components- FRPs are epigenetically inactivated in colon cancer (Suzuki et
al, 2004)
• Lgs/Bcl-9 implicated in B cell malignancies (Kramps, 2002).Frat/GBP is activated by a proviral
insertion in mouse lymphomas (Jonkers, 1997).Many other signalling components have been
found upregulated in cancer cells.Eg, Dsh has been found upregulated in lung cancer and
mesotheliomas
 • Wnt5a- In some cellular contexts Wnt5a acts as an antagonist of Wnt signalling.It seems to do so
independently of APC function but does require GSK3.Leads to direct degradation of -catenin
14. Wnt signalling may make a significant contribution to different types of cancers, the precise
contribution it makes may differ between tumour types.

LRP5 and Bone Density

• Human LRP5 and 6 had previously been cloned due to homology with LDL receptors before their role
in Wnt signalling was appreciated
• Work has found mutations in LRP5 in individuals with both low and high bone mass
• Loss of function of LRP5 is associated with low bone density, gain of function of LRP5 is associated
with high bone density
Frizzled, LRP5 and FEVR
• Familial Exudative Vitreoretinopathy (FEVR): inherited blinding disorder of the retinal vascular
system
• Mutations in Frizzled 4 and LRP5 have been shown to be invovled
• Wnt signalling thus required for vascularisation of the eye

Mutations in LRP5 Leading to FEVR

Therapy by Targeting Wnt Signalling Components

• There are many levels of control of the Wnt signalling pathway
• Any of these could be targeted for therapy
• Knock down transcript levels with antisense or RNAi
• Knock down/inactivate proteins by netralising antibodies
Targeting Wnt Signalling: Extracellular
• Wnts have been targetted with antibodies
• FRPs or Dkks could be used to inhibit
• Dkk-3 expression in non-small cell lung carcinoma cell inhibited cell growth (Tsuji et al 2001)
Dkk-3 and dominant-negative LRP5 expression in Saos-2 cells reduced invasion capacity and motility
Targeting Wnt Signalling: Cytoplasmic
• Introduction of wild-type Axin-1 induced apoptosis in hepatocellular and colorecetal cancers (Satoh, et
al 2000)
• A small molecule inhibitor of Dsh physical interactions has been described that down regulates
signalling in culture and increases apoptosis of cancer cell lines
Targeting Wnt Signalling: β-Catenin Expression and Degradation
• Antisense against β-Catenin decreased expression and led to tumour regression (Luu et al 2004)
• Introduction of a mutant F-box protein (part of proteolytic machinery) led to increased targetting for
degredation of free (but not cadherin bound) β-Catenin (Liu, et al, 2004)
• β-Catenin degredation has been found to be enhanced by several natural compounds
Targetting Wnt Signalling: Nucleus
• Structural details of the β-Catenin/Tcf complex highlight possibility of developing drugs to inhibit such
interactions
• 7000 natural compounds screened for inhibition of interaction (lepourcelet et al, 2004)
• Several found that also reduce β-Catenin-dependent activities