Wingless flies

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Wingless flies
Eyeless fish
Brainy mice
Stem Cells
Two-headed tadpole
Wnt Signaling
Deaf Mice
Human colon Cancer
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What is Signal Transduction ?
Extracellular Signal
Cell Membrane
Cellular Effects
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20 signaling pathways (Gerhart, 1999)
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Despite the large number of receptors, ligands,
and down-stream targets, the number of signal
transduction pathways in metazoans is relatively
small, auguably less than 20. (Gerhart, 1999)
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Role of Wnt signaling in cancer and development
Development Primary body axis Gastrulation Pattern
ing the brain Cardiogenesis Fat formation Bone
marrow differentiation Stem cell maintenance
Cancer Colorectal cancer B-cell
lymphoma Hepatocellular carcinoma Melanoma Endomet
rial cancer Ovarian cancer Prostate cancer
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How do we study the Wnt signal transduction
pathway?

• Tissue culture-TopFlash (canonical signaling)
• Xenopus embryos and explants
• Mouse genetics
• Drosophila genetics
• Zebrafish embryo injections and genetics

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Much of our understanding of the Wnt pathway has
come from a convergence of studies from people
interested in cancer, development, and signal
transduction.
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Many Tumors Induced by the Mouse Mammary Tumor
Virus Contains a Provirus Integrated in the Same
Region of the Host Genome. Nusse , R. and
Varmus, H., Cell. 1982. Vol. 31, 99-109
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• Oncogenesis by insertional mutagenesis
• Virus, once inserted into the host genome
  adjacent to c-onc, its regulatory sequences
  stimulate host oncogenes
• U3 regions of viral genome are known as long term
  repeats or LTRs
• U3 contains all the promoter information
  necessary to begin transcription of c-onc so long
  as virus is integrated in proximity to cellular
  oncogene

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The Drosophila homolog of the mouse mammary
oncogene int-1 is identical to the segment
polarity gene wingless. Rijsewijk F, Schuermann
M, Wagenaar E, Parren P, Weigel D, Nusse R.
Cell. 1987 Vol. 50, 649-57
Phenocopies induced with antisense RNA identify
the wingless gene. Cabrera CV, Alonso MC,
Johnston P, Phillips RG, Lawrence PA. Cell.
1987. Vol. 50, 659-63.
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wg-
wt
wg- HSwg
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int1 wingless
wingless int1 wnt
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Hans Spemann Hilde Mangold
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Whats the significance? Revealed for the 1st
time the ability of one tissue to organize
another tissue. It established the concept of
inductive processes. --host cells CHANGED their
fate to tissues they normally never form, due
to an inductive center being placed adjacent
to them.
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Ectopic expression of the proto-oncogene int-1 in
Xenopus embryos leads to duplication of the
embryonic axis. McMahon AP, Moon RT. Cell. 1989
Sep 2258(6)1075-84.
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Uninjected
Int-1 injected
Xenopus laevis
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Cancer Research (Oncogenesis)
Drosophila Development (Segment polarity)
Xenopus Development (Mesoderm inductions)
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The Drosophila segment polarity gene dishevelled
encodes a novel protein required for response to
the wingless signal. Klingensmith J, Nusse R,
Perrimon N. Genes Dev. 1994 Jan8(1)118-30
Autonomous requirements for the segment polarity
gene armadillo during Drosophila
embryogenesis. Wieschaus E, Riggleman R. Cell.
1987 Apr 2449(2)177-84.
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Positive regulators of Wnt signaling
Wnt
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WT
Wg- (Decreased Wnt signaling)
Zw3- (Gsk3-) (Increased Wnt signaling)
Wingless signaling acts through zeste-white 3,
the Drosophila homolog of glycogen synthase
kinase-3, to regulate engrailed and establish
cell fate. Siegfried E, Chou TB, Perrimon
N. Cell. 1992 Vol. 71, 1167-79.
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V
D
D
Glycogen synthase kinase-3 and dorsoventral
patterning in Xenopus embryos. He X,
Saint-Jeannet JP, Woodgett JR, Varmus HE, Dawid
IB. Nature 1995 Vol. 18, 253-622
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The effects of a lethal mutation responsible for
duplications and twinning in mouse embryos. J.
Exp. Zool. 1949 Vol. 110, 4776. Gluecksohn-Schoen
heimer, S.
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The mouse Fused locus encodes Axin, an inhibitor
of the Wnt signaling pathway that regulates
embryonic axis formation. Zeng L, Fagotto F,
Zhang T, Hsu W, Vasicek TJ, Perry WL 3rd, Lee JJ,
Tilghman SM, Gumbiner BM, Costantini F. Cell.
1997 Vol. 90, 181-92.
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Epistasis experiments indicate that positive and
negative regulators act upstream or at the level
of ?-catenin
Positive regulators
Negative regulators
GSK3
Dsh
Axin
-

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What is the role of ?-catenin?
Previously shown to be part of a complex of
protein that links Cadherins to the cytoskeleton
Microfilament
Cadherin
?-catenin
?-actinin
Vinculin
?-catenin
Plasma-membrane
Catenin chain in Greek
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Epistasis experiments indicate that positive and
negative regulators act upstream or at the level
of ?-catenin
Positive regulators
Negative regulators
GSK3
Dsh
Axin
-

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?-catenin staining shows dorsal nuclear
localization (dorsal activation)
Dorsal
Ventral
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Functional interaction of ?-catenin with the
transcription factor LEF-1. Behrens J, von Kries
JP, Kuhl M, Bruhn L, Wedlich D, Grosschedl R,
Birchmeier W. Nature. 1996 Vol. 382, 638-42.
Nuclear localization of ?-catenin by interaction
with transcription factor LEF-1. Huber O, Korn R,
McLaughlin J, Ohsugi M, Herrmann BG, Kemler
R. Mech Dev. 1996 Vol. 59, 3-10.
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Insight from Drosophila

• Wg RNA is present in a segmental pattern
• 2) Amadillo RNA is uniform and ubiquitous

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Wg RNA
Arm (?-catenin) Protein
Spatial expression of the Drosophila segment
polarity gene armadillo is posttranscriptionally
regulated by wingless. Riggleman B, Schedl P,
Wieschaus E. Cell. 1990 Vol. 63, 549-60.
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Conclusion Wnt signaling is associated with the
stabilization of ?-catenin
What is the role of ?-catenin degradation in Wnt
signaling?
Wnt
?-catenin
Degraded ?-catenin
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Insight from studies of colorectal cancer Role
of the Adenomatous Polyposis Coli protein (APC)
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APC CoIP with ?-Catenin
1801-Anti p53 CF11-Anti APC HECD-1- Anti
E-cadherin
Association of the APC tumor suppressor protein
with catenins. Su LK, Vogelstein B, Kinzler
KW. Science. 1993 Vol. 262, 1734-7.
Association of the APC gene product with
beta-catenin. Rubinfeld B, Souza B, Albert I,
Muller O, Chamberlain SH, Masiarz FR, Munemitsu
S, Polakis P. Science. 1993 Vol. 262,1731-4.
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APC promotes the degradation of ?-catenin
Regulation of intracellular ?-catenin levels by
the adenomatous polyposis coli (APC)
tumor-suppressor protein. Munemitsu S, Albert I,
Souza B, Rubinfeld B, Polakis P. Proc Natl Acad
Sci U S A. 1995 Mar 2892(7)3046-50.
Loss of APC (observed in a majority of colorectal
cancers and malignant melanomas) lead to elevated
?-catenin levels.
Constitutive transcriptional activation by a
?-catenin-Tcf complex in APC-/- colon
carcinoma. Korinek V, Barker N, Morin PJ, van
Wichen D, de Weger R, Kinzler KW, Vogelstein B,
Clevers H. Science. 1997 Mar 21275(5307)1784-7.
Activation of beta-catenin-Tcf signaling in colon
cancer by mutations in beta-catenin or APC. Morin
PJ, Sparks AB, Korinek V, Barker N, Clevers H,
Vogelstein B, Kinzler KW. Science. 1997 Mar
21275(5307)1787-90.
Stabilization of beta-catenin by genetic defects
in melanoma cell lines. Rubinfeld B, Robbins P,
El-Gamil M, Albert I, Porfiri E, Polakis P.
Science. 1997 Mar 21275(5307)1790-2.
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APC is a negative regulators of
?-catenin-mediated transcription and stability
Positive regulators
Negative regulators
GSK3
Dsh
Axin
-

? Genes regulated by ?-catenin-TCF
Transcription
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Positive Regulation of the Wnt Pathway
Wnt
Cell membrane
Degraded ?-catenin
Transcription of Target Genes
?-catenin
Tcf
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What are the roles of the negative regulators?
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APC, GSK3, and Axin promote the degradation of
?-catenin
GSK3
Axin
b-catenin
Degraded b-catenin
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Key feature of canonical Wnt signaling
Regulated degradation of ?-catenin
P
?-catenin
P
P
Ub
Ub
GSK3
Ub
Ub
Ub
Degraded ?-catenin
Axin
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Key feature of canonical Wnt signaling
Regulated degradation of ?-catenin
P
?-catenin
P
P
Ub
Ub
GSK3
Ub
Ub
Ub
Degraded ?-catenin
Axin
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?-catenin is ubiquitinated by the SCF ubiquitin
ligase complex
Ub
Ub
Ub
Ub
Ub
Ub
Ub
SCF ubiquitin ligase
APC
Ub
Ub
Ub
Ub
P
Ub
b-catenin
P
P
Ub
Ub
P
Ub
b-catenin
SCF Skp1, Cullin, and F-box (?-TRCP) E3
ubiquitin ligase
Conjugated b-catenin
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2000
?
?
P
P
P
P
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LRP-LDL Receptor Related Proteins
Strickland et al. (2002)
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Role of LRP5/6 in Wnt signaling
An LDL-receptor-related protein mediates Wnt
signalling in mice. (2000) Pinson KI, Brennan J,
Monkley S, Avery BJ, Skarnes WC. Nature 407,
535-8.
Arrow encodes an LDL-receptor-related protein
essential for Wingless signalling. (2000) Wehrli
M, Dougan ST, Caldwell K, O'Keefe L, Schwartz S,
Vaizel-Ohayon D, Schejter E, Tomlinson A, DiNardo
S. Nature 407, 527-30.
LDL-receptor-related proteins in Wnt signal
transduction. (2000) Tamai K, Semenov M, Kato Y,
Spokony R, Liu C, Katsuyama Y, Hess F,
Saint-Jeannet JP, He X. Nature 407, 530-5. 
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Screen for recessive lethals by gene trap
insertion in cell-surface proteins
An LDL-receptor-related protein mediates Wnt
signalling in mice. (2000) Pinson KI, Brennan J,
Monkley S, Avery BJ, Skarnes WC. Nature 407,
535-8.
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First 321 amino acids of LRP6 upstream of the
reporter
Developmental defects were similar to mouse
mutations in Wnt-3a, Wnt-1, and Wnt-7a
Wnt-3a-like
Vertebrates caudal to the lumbar regions are
absent
An LDL-receptor-related protein mediates Wnt
signalling in mice. (2000) Pinson KI, Brennan J,
Monkley S, Avery BJ, Skarnes WC. Nature 407,
535-8.
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WT
LRP6 mutant
isthmus
Wnt-1 phenotype-mid/hindbrain defects
Caudal midbrain missing
An LDL-receptor-related protein mediates Wnt
signalling in mice. (2000) Pinson KI, Brennan J,
Monkley S, Avery BJ, Skarnes WC. Nature 407,
535-8.
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LRP6 mutant
Wt
Wnt7a- loss of posterior digits
An LDL-receptor-related protein mediates Wnt
signalling in mice. (2000) Pinson KI, Brennan J,
Monkley S, Avery BJ, Skarnes WC. Nature 407,
535-8.
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WT
arrnull
Arrow encodes an LDL-receptor-related protein
essential for Wingless signalling. (2000) Wehrli
M, Dougan ST, Caldwell K, O'Keefe L, Schwartz S,
Vaizel-Ohayon D, Schejter E, Tomlinson A, DiNardo
S. Nature 407, 527-30.
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LDL-receptor-related proteins in Wnt signal
transduction. (2000) Tamai K, Semenov M, Kato Y,
Spokony R, Liu C, Katsuyama Y, Hess F,
Saint-Jeannet JP, He X. Nature 407, 530-5. 
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Conclusion LRP5/6/Arrow plays a role in
transmitting a Wnt signal
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Basic mechanism of the Wnt signal transduction
pathway
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Secreted negative regulators of the Wnt pathway
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DKK-1 is a Wnt inhibitor that targets LRP5/6
Dkk
LRP5/6
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Other Wnt antagonists and related proteins
Kawano, Y. et al. J Cell Sci 20031162627-2634
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Mechanism of regulation of Wnt signaling by
antagonists
Kawano, Y. et al. J Cell Sci 20031162627-2634
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The Wnt Pathway is a three-headed beast
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The Wnt Pathway is a three-headed beast
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Examples of planar cell polarity (PCP) mutants in
Drosophilla
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Non-canonical Wnt signaling regulates Convergent
Extension Movements
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Dsh control morphogenetic movement during Xenopus
development
Xdd injections
(Dominant negative for the canonical pathway)
Analysis of Dishevelled signalling pathways
during Xenopus development. Curr Biol. 1996, 6
1456-67. Sokol SY.
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Stereocillia in the organ of Corti
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Emergent oncogenic (non RTK) pathways
TGF-?
Cancer (Stem?) Cell Viability
Wnts
Notch Ligands
Hedgehog
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CANCER CELL INHIBITORS
TGF-?
Cancer Stem Cell Death
Rx
Rx
Wnts
Rx
Notch Ligands
Rx
Hedgehog
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Wnt signaling is active in the early Xenopus
embryo
Collect Eggs
Cytoplasm
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Validation of hits
Perturb axis formation in Xenopus embryos
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WS30 ventralizes Xenopus embryos in dose
dependent manner
vehicle
WS30
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WS30 is a Potent Wnt Inhibitor
Wnt Activity Inhibition Assay
EC5050nM
EC50 50 nM
Wnt Activity
WS30 concentration (M)
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Effects of WS30 on MDA-MB-231 breast cancer cells
WS30
vehicle
WS30 is active on both breast and colon (SW480
SW620) cancer cells