Wnt Discovery 1. Integration Of Mouse Mammary Tumour Virus (mmtv)

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 • Epistasis: the loss of one gene masks the effect of the loss of another • Double mutant-when 1 mutant masks the phenotype of the other. •

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Wnts Extracllular Antagonists: Frzbs, Dikkopf Receptors: Frizzled, Arrow,LRP5/6 Cytoplasmic: Dsh, APC, Axin, ZW3, Arm Nuclear: Groucho, TCF, Arm, Pan, Lgl

WNT PROTEINS

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1. The Wnt proteins are a family of small (39–46 kDa) lipid modified secreted glycoproteins (Made of 350-400 a.as). They contain 23–24 cysteine residues with highly conserved spacing Wnt proteins are ligands for the Frizzled family of seven-pass transmembrane receptors. 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 • • •

Product of the mouse fused locus Antagonises Wnt signalling Regulator of G-Protein Signalling (RGS) domain and DIX domain

APC • • • • •

Adenomatous Polyposis Coli Major tumour suppressor Multiple cellular roles: also regulates microtubules Huge protein, many domains and protein interactions Antagonises Wnt Signalling

Zest-white3/Shaggy/GSK3 • Glycogen Synthase Kinase 3 (GSK3) • A sereine/threonine kinase • Long known to phosphorylate Armadillo, now also known to phosphorylate APC, Axin and Arr • Antagonises Wnt signalling •

Armadillo/-Catenin • Roles in cell adhesion as part of the cadherin/catenin junctional complexes • Key mediator of Wnt/Wg signalling • In absence of signalling cytoplasmic levels are low, during signalling levels are increased • Activates transcription but can’t bind DNA

TCF and Groucho • T-cell factor, can bind DNA but can’t activate transcription • In the absence of signalling they repress transcription of target genes in concert with groucho • In the presence of signalling b-catenin displaces Groucho from TCF and turns on transcription at the same promotors

Minimal Physical Interactions • Wnt: Fz and Arr • Fz: Dsh • Arr: Axin • Dsh: Axin and GBP • Axin: APC: -catenin • GSK3: GBP, Axin and -catenin • -catenin: TCF The Destruction Complex • When Wnt signalling is inactive, APC and Axin act as scaffolding proteins to bring GSK3 and other kinases into proximity with -catenin • Phosphorylation of -catenin tags it for destruction by the proteosome

Pygopus and Legless/Bcl9 • Legless/Bcl9 links b-catenin to Pygopus which is required for the transcription of Wnt responsive genes

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.

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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. 6.

This results in accumulation of cytosolic b-catenin. β- Catenin protein is normally found at low levels in the cytoplasm but activation of Wnt signalling will raise βcatenin levels. 7. The β-catenin, stabilized under Phosphorylation will then translocate into the nucleus and bind to members of the Tcell 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. 2. 3. 4. 5.

β-catenin is attached to the membrane by E-cadherin and α-actin. A complex of Axin, APC and the serine-threonine kinase GSK3b binds and phosphorylates cytoplasmic b-catenin. This triggers ubiquitination by an E3 ligase complex containing b-TrCP, resulting in proteolytic degradation of bcatenin. Phosphorylated b-catenin is recognized by b-transducin repeat-containing protein (b-TrCP) and degraded by the proteaosome. 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.

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secreted frizzled-related proteins (sFRPs) (act as Wnt agonists) binds with Wnt ligand and prevent it from binding with its receptor. Wnt-inhibitory factor-1 (WIF1)contains conserved WIF domain with five epidermal growth factor (EGF)-like repeats. 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. 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- βCatenin 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 of 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 halflife of less than 60 minutes. 2. Axin forms a complex with βCatenin/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 pof Axin and APC and PP2A with B56 subunit. 5. Within the complex, GSKβ p- N-terminus of βCatenin (has regonition site for GSKβ b/w a.as 3345 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 3237 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,Rbx1,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 Activation of Wnt Signalling (the ‘‘On’’ Switches) Cytoplasmic β-catenin translocate to the nucleus 1. β-Catenin competes with Groucho for binding with the LEF/TCF proteins 2. The LEF/TCF proteins allow β-catenin to bind to the DNA. 3. Thus forms a large complex of molecules that allows specific target genes to be transcribed. They are  essential cofactors( pygopus and Bcl-9/legless)  proteins (such as p300/Creb binding protein (CBP)  Pontin52 4. Specific target genes to be transcribed by various mechanisms - Histone modification of chromatin -acting as bridges between β-catenin and the transcriptional machinery. 5. β-catenin targets genes are as c-myc, c-Jun, and Sox9. 6. In turn,these genes alter the expression of their own target genes.

Inhibition of Wnt Signalling (the ‘‘Off’’ Switches) Cytoplasmic β-catenin translocate to the nucleus 1. Without Wnt ligand stimulation, it is insufficient to activate target gene transcription. 2. β-Catenin cannot bind to DNA at the consensus motif (A/T)(A/T)CAA(A/T)G to activate transcription. 3. It complex with members of the LEF/TCF family of high mobility group (HMG) proteins. 4. This provides a DNA binding domain for β-catenin. 5. The LEF/TCF family consists of four proteins (LEF-1, TCF1, TCF3, and TCF4) 6. In the absence of nuclear βcatenin, the LEF/TCF proteins are found in complex with transcriptional repressors such as

Feedback Loops and Other Signalling Pathways Positive feedback loops that enhance the Wnt signalling pathway. 1.

2. 3. 4.

5.

Increased levels of β-catenin can increase Lef-1 splice variant(favours further β-catenin-mediated transcription ) Increased levels of CKII after Wnt stimulation can stabilize β-catenin protein. Integrin signalling, can cause nuclear localization of β-catenin Few that activates Wnt signalling by inhibiting GSK3β insulin-like growth factor (IGF) signalling Kras signalling ( to facilitate stabilization of βcatenin) EBV STAT3 signalling can stimulate nuclear translocation of β-catenin.

Groucho and CtBP . Histone deacetylases (transcriptional repressors), are also recruited to the complex, to maintain gene repression. 8. Chibby and Sox proteins ( HMGproteins), competes with LEF/TCF proteins for β-catenin. 9. Duplin and ICAT ( β-catenin binding proteins) prevents formation of β-catenin/TCF complexes. 10. β-catenin/TCF complexes binding to DNA is inhibited by p- of TCFs by Nemo-like kinase (Nlk). 7.

Feedback Loops and Other Signalling Pathways 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. 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 – Bcell Lymphoma 1 -catenin Activation- Loss of phosphorylation sites in cancer render it refractory to destruction. 1.

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Mutations in Frizzled and LRP5 surface receptors, and other Wnt proteins as it is constant “On” state Molecules that inhibit Wnt signalling are inactivated through either loss-of-function mutations or epigenetic silencing. 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 When APC IS lost , destruction complex can’t form, -catenin isn’t phophorylated and so constitutively activates transcription Mutations in APC and AXIN1/2 (are integral to the β-catenin phosphorylation complex) cause destabilization of the complex. Somatic APC mutations will result in loss of the Axin binding sites.This shows that loss of βcatenin regulation is the major selective drive for APC mutations. 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. Many cancers have CTNNB1 mutations. 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) Mutation of the β-catenin gene alone is sufficient for adenoma development in the intestine. APC mutations are rarely colon. 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

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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