“understandably, the search for these cross connections ... · ra weinberg, the biology of...
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“Understandably, the search for these cross connections has been largely postponed until the operations of each primary pathway are elucidated.” RA Weinberg, The Biology of Cancer, 2007, p202.
Genetic Variation at the Low Density Lipoprotein Receptor‐Related Protein 5 (LRP5) Locus Modulates Wnt Signaling and the Relationship of Physical Activity with Bone Mineral Density in Men
Kiel et al, Bone, 2007, 40(3): 587–596.
WT
alle
le
Hap
loty
pe1
Hap
loty
pe2
G-T
-G-T
A-T
-G-T
WT
alle
le
TOP
flash
activ
ity in
HE
K29
3 ce
llstra
nsfe
cted
with
LR
P5
hapl
otyp
es
Wnt3a - + - + - +
… “the TT genotype was associated with lower BMD in men with higher physical activity scores, conversely with higher BMD in men with lower physical activity scores”.
essential & known components
non-essential known components
“novel” components
MB Major et al, Science Signaling, 2008, 1(45): ra12
BW Miller et al, Mol. Sys. Biol., 2009, (5):315
Cytoscape visualization of the Wnt pathway from cancer.cellmap.org (netpath.org data)
GSK3β
APC
Axin1
Dvl2
Wnt Planar Cell Polarity Pathway
KEGG Canonical Wnt signaling
KEGG colorectal cancer pathway
netpath.org
WikiPathways
APC
APC
PORCWLS
free Wnt(short range)
complexed Wnt(long range)
Flottillin-2
retromer
GPC3
Morphogendiffusion profile
ligand processing & delivery
signal transduction
regulation of transcription
degradation complex
Wnt ligand processing and secretion
heparan sulfate proteoglycans: GPC3
PORCWLS
Flottillin-2
retromer
complexed Wnt(long range)
free Wnt(short range)
GPC
3
Morphogendiffusion profile
Xin
g et
al,
Gen
es &
Dev
. 200
3 17
: 275
3-27
64
β-catenin degradation in the absence of Wnt signaling
Axin:Apc:Gsk3β:Diversin:CKIα/ε:PP2A:WTX+
β-catenin
Axin:Apc:Gsk3β:Diversin:CKIα/ε:PP2A:WTX:β-catenin|p
Axin:Apc:Gsk3β:Diversin:CKIα/ε:PP2A:WTX
+
β-cateninβ-Trcp, Skp1, Cul1, Rbx
recy
cle
|p
|p
Wnt signaling – (1) Receptor Complex Formation & signal amplification
+ Lrp+ Fz
LFzW
+ LFzWDFrGαβγ
Wnt ------------------PP1 , PP2A,B
CK1ε
(CK1ε)p
+ Frat-1
Dvl
(Dvl)pp (Dvl)pp:Fr
LFzWDFr + Ck1γ
+ Axin:GSK3LFzWD(AGC)Fr LFWD(AGC)
PP
destruction complex
freeβ-catAPC
to nucleus
MACF1
+
Axin:GSK3
LFWD(AGC)
PP
Model
destruction complex
Wnt Axin,Gsk3β
β-catAPC
Receptorcomplex
n
Wnt signaling – (2) Receptor Aggregation & Clustering
Lrp aggregation
PIP5K1A, PI4KIIα
LFzWDFr(AGC)PIP2
L FWD(AGC)
PP m
L FWD (AGC)
PP m
L FWD (AGC)
PP mq
n.mn
n.m
L FWD (AGC)
PP mq
n.m.k
n
Overall Model
destruction complex
Wnt A,G,C
β-catAPC
Signalamplification
Receptoraggregation
Receptorclustering
PP
Dvlmediatedreceptorclustering
In the nucleus
+ phosphorylation of Tcf and Tle by Ck1 and Ck2
Wntnuclear(APC)P
Cytoplasmic β-cat
Nuclear β-cat
transcription
repression
Histone modifications
SET1 H3K4me
β-cat:Pygo bind H3K4me, dissociate from TCF
CtBP
KRAS/RAF1/RAC1
BCL-9, Pygode
laye
d / s
low CBP, P300, TRRAP,
SWI/SNF, BRG1,Mediator, COMPASS
PAF1, HRPT2, MLL1/2
export /
degradation RanBP3, RanGTPβ-Trcp, Skp1
Cul1, Rbx
HDAC-1,SHARP,TLE, CtIP,CtBP1/2X
Data: BW Miller et al, Molecular Systems Biology, 2009, (5):315
My comments:
Less DVL sequestration
Canonical W
nt pathway genes e
xtracte
d from Moon lab gen
ta notome-wide screen (M
ichael M
ajor, Jason Berndt et al)
Unpublished da
shown.
Kofahl & Wolf, Biochem. Soc. Trans., 2010, (38):1281–1285;
Kruger & Heinrich, Genome Informatics, 2004, 15(1): 138-148.Full model : 15 ODEs, 31 parameters Reduced : 7 ODEs, 8 algebraic equations, 19 parameters(Same model as Lee et al, 2003)
Bold face = measured in Xenopus egg
extracts
Italics = estimated
3 decimal points!
Lee et al, PLoS Biology, 2003, 1(1):116-123.
guess-timatedfrom above
Oksana Schaeffer-Tymchyshyn, PhD Thesis, Dept CS, U. Birmingham, 2008.
46 rate equations, modeled with ODE’s and also using stochastic p-calculus
(simian) COS cells + in-vitro
Xenopus egg extractsSW480 + COS + in-vitroin-vitro
in-vitro
(mouse) C57MG cells
in-vitro
(drosophila) S2 cells(human) 3T3 cells
(mouse) C57MG cells
in-vitro(human) MB231 cells
(human) MB231 cellsXenopus egg extracts
(simian) COS cells + in-vitro
Cho, Baek & Sung, FEBS Letters, 2006, (580):3665–70.
18 “elementary” reactions
Based on: Lee, Salic, Kruger, Heinrich, Kirschner, PLoS Biology, 2003, (1):E10.
For a mutant with n missing sites:
KD(n) = cn * KD(WT)
Cho, Baek & Sung, FEBS Letters, 2006, (580):3665–70.
Immunoblots in engineered
mouse F9 cells
Yokoyama et al, Journal of Molecular Signaling 2007, (2):11
Kinetics of signalosome formation in HeLa cells
Bilic
eta
l, Sc
ienc
e, 2
007,
(316
):161
9–16
22.
Axin=green, Lrp6=red, arrows=membrane-associated co-aggregates
Mirams, Byrne & King, J Math Biol. 2010,60(1):131-60.
1-equation formal reduction of Lee et al (2003) model (for Xenopus):
ββ β
Where:
Mirams’ extension to include Axin2 feedback
ββ
ββ
β
Qualitative predictions using Miram’s 1-equation model Wnt dose response
β−catenin
Wnt
Bie
chel
eet
al,
Che
mis
try &
Bio
logy
, 200
7, (1
7):1
177–
1182
.
log (Wnt)
β−catenin
Predictions using Miram’s 1-equation model
GSK3 RNAi
DVL RNAi
contradicts Moon lab RNAi data & Miller et al, 2009, MSB, (5):315
X
X
√√
β−catenin time course
~ 8
hour
s
√GSK3 OE
APC RNAiAPC OE
√DVL OE
Wnt=1
Predictions using Miram’s 1-equation model
GSK3 RNAicontradicts Miller et al, 2009, MSB, (5):315
XGSK3 OE
APC RNAiAPC OE
√DVL OE
Wnt=0
β−catenin time course
Goentoro and Kirschner’s 1-equation model, Molecular Cell, 2009, (36):872–884
Wnt+Wnt
β-cat β-cat− β-cat
β-cat
APC-mediated degradation of Axin
steady state β-catenin concentration is a saturating function of APC & GSK3β levels
stea
dy s
tate
β-c
aten
in
GSK3β
GSKβ OE
X
stea
dy s
tate
β-c
aten
in
APC
APC RNAi
X
Power Law approximate kinetics – Saturable and Cooperative formalism
condition1 condition2 condition3
30 p
aram
eter
s
Input X1 X2v1 v2v3
v5
X3 v4
X4 v6
Sorribas et al, Biotechnology and Bioengineering, 2007, 97(5):1259-1277.
( )GinputfionmaxExpresstimeScaledtdG
Ginputfkkk
dtdGGkinputfk
dtdG
d
tddt
−=
−=−=
)(..
)(. .)(.
:lyequivalent
:lyequivalent
maxExpression1
maxExpression2
timeScale1
timeScale1 G (A
.U.)
input
Time (A.U.)timeScale2
inputper threshold1 inputs offunction logic and sevaluation node theoforder in the encoded is
set is :models logicIn
⇒=
=
f(input) timeScale
1 ionmaxExpress
RNAi RNAi OE OE
– Wnt + Wnt – Wnt + Wnt
Ub Core UBE2M, CUL1, DUSP18, βTRCP
SKP1A ↑lower β-cat degradation
↑ ↑
APC ↑AXIN 1/2 ↑ ↑ M ↑ M ↓ M
SIAH1 ↑ (degrades β-cat)
SMURF1/2 ↓ M (degrades Axin) ↓ (activates PCP)
TCF/LEF1 ↓ ↑ ↑LRPs/FZs ↓
Wnt4/5B/11 ↓ M
sFRP2 ↓ M
WIF1 ↑FRAT1/GNAi1 ↓ ↓ ↑ ↑
PP1γ ↓ (as Axin OE)
DVL1/2/3 ↑ X (see idea) Miram’s ↓ ↑ Miram’s ↑ ↑ Miram’s ↑NKD1 ↓ M ↓ M
GSK3α/β ↓ X GSK3β titration? α↓ ↓β M Miram’s ↑ ↓ M Miram’s ↓CSNK2A1/2 ↑ (β-cat degradation) ↑ (β-cat degradation)
CSNK1ε ↓ (signalosome) ↑ (signalosome) ↑ (signalosome)
CSNK1δ ↑ (in DC) ↓ (in signalosome) ↑ (in signalosome) ↑ (in signalosome)
CTNNB1 ↓ ↓ ↑ ↑
proteasomal degradation of Axin and APC
CSK2 is constitutively active
SMURF2 binds Axin and destabilizes it
HEK293T
HE
K29
3T
Kim & Jho, Journal of Biological Chemistry, 2010, 285(47):36420–36426.
Narimatsu et al, Cell, 2009, (137):295–307.
(in mice)
HEK293 cells over‐expressing Dvl‐2 (green)blue=nuclear stain
These cytoplasmic Dvl aggregates are not required for Wnt/β‐catenin signaling
Sm
alle
y et
al J
ourn
al o
f
red=F-actin
Cel
l Sci
ence
, 200
5, (1
18):5
279-
5289
HEK293s with Dvl‐2 (green) at ~ physiological level
Hypothesis: Over-expressed Dvl recruits Axin out of the destruction complex β-catenin signaling(strong inter-DIX-domain interaction between Dvl and Axin)
A parsimonious model of canonical Wnt 24-48hr response in HEK293
Nkd1
Axin1+Axin2Dvl(with GSK3 etc)
Destruction Complex
Wnt signalosome
β-catenin
NKD1AXIN2
X
Fzd’s sFRP’sCanonical Wnt’s
X
non‐canonical Wnt’sβ-catenin
(Adherent) A375 cells expressing Wnt3a
Wnt3a gradient due to flow
Cimetta et al, Lab on a Chip, 2010, (10):3277–3283.
Reported graded response to ligand concentration in canonical Wnt signaling
minutesunst
imua
ted
(60ng/ml rWnt3a)
(β-catenin firefly luciferase reporter in HEK293T cells)
Naik and Piwnica-Worms, PNAS, 2007, 104(44):17465–17470.
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Out
put (
A.U
.)
Normally distributed thresholds
mean=0.5, std=0.05Ave
rage
of 2
000
step
func
tions
log (x)
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
1.0
Out
put (
A.U
.)A
n ex
ampl
e st
ep fu
nctio
n
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
Out
put (
A.U
.)A
n ex
ampl
e M
M fu
nctio
n
0.0 0.2 0.4 0.6 0.8 1.0
0.0
0.2
0.4
0.6
0.8
Out
put (
A.U
.)
Sum of 2000 MM functions Normally distributed KMs, mean=0.1, std=0.01
Ave
rage
of 2
000
MM
func
tions
log (x)
β-catCk2 TLE1
in C2C12 mouse myoblast cells
S W
ang
& K
A J
ones
Minimal model
Cytoplasmic β-cat nuclear(APC)P
Wnt
Nuclear β-cat
transcription
repression
Histone modifications
SET1 H3K4me
β-cat:Pygo bind H3K4me, dissociate from TCF
export /
CtBP
dela
yed
/ slo
wdegradation
X
YX
in
X
Y
in=low in=high
4321
4
21
,,
...1
.3
..1
.
kkkk
ykxkink
inkdtdy
xkink
inkdtdx
xj
i
<
−++
=
−+
=
(x slower than Y)
Nkd1 RNAi ↓ β-catenin
Nkd1 OE ↓ β-catenin
Wnt
β-ca
teni
n
Dvl
β-ca
teni
n
Axin1
β-ca
teni
n
GSK3β
β-ca
teni
n
Nkd1l
β-ca
teni
n
Axin2
β-ca
teni
n
Predicted steady state β-catenin levels
Dashed red lines indicate model’s un-optimized operating point
Gujral & MacBeath, PLoS ONE, 2010, 5(4):e10024.
HEK293
Nkd1:Dvl3 aggregation
Nkd1 Dvl3
HEK293 cells+ rWnt3a
Nkd1
HEK293 cellsNkd1:Dvl3 aggregation (Guo et al)
Guo et al, PLoS ONE, 2009, 4(11):e7982.
Nkd1 localizes to the cell membrane
Nkd1 mutations blocking Dvl interaction increase β-catenin in HEK293 cellsG. J. Lau, MS Thesis, Dept Biochem, U Toronto, 2008
Nkd1 interacts with Axin1B
W M
iller e
t al,
Mol
ecul
ar S
yste
ms
Bio
logy
, 200
9, (
5):3
15
(Over)expressed Nkd1: Dvl sequestration β-catenin/TCF ↓Nkd1 RNAi: Axin1 sequestration ↓ β-catenin/TCF ↓
Gar
net J
ean
Lau,
MS
The
sis,
Dep
t Bio
che
HEK293
m, U
Tor
onto
, 200
8
HEK293
HEK293
(Over)expressed Nkd1: Dvl sequestration β-catenin/TCF ↓Nkd1 RNAi: Axin1 sequestration ↓ β-catenin/TCF ↓
WT NKD1 Nkd1
Axin1+Axin2(+ etc)
Dvl
signalosomeWnt Destruction Complex
β-catenin
NKD1AXIN2
Nkd1
Axin1+Axin2
signalosome
Dvl
Destruction Complex
β‐catenin
NKD1AXIN2
Wnt
Nkd1
Axin1+Axin2
signalosome
Dvl
Destruction Complex
β‐catenin
NKD1AXIN2
Wnt
OE RNAi
Wnt4 inhibits canonical Wnt signaling by redirecting β-catenin to the cell membrane
HEK293T cells transfected with Wnt3a or Wnt4
Bernard et al, Biology of the Cell, 2008, (100):167–177
sFRP2 enhances Wnt signaling & β-catenin nuclearization
HEK293 cells
Von Marschal & Fisher, Biochemical and Biophysical Research Communications, 2010, (400):299–304.
sFRP2 up-regulates Wnt target genes
mR
NA
/ co
ntro
l
HEK293 cells
HEK293 cells
Von Marschal & Fisher, Biochemical and Biophysical Research Communications, 2010, (400):299–304.
But are expressed in response to canonical Wnt
Gujral & MacBeath, PLoS ONE, 2010, 5(4):e10024.
HEK293 cells + 24 hours 200ng/ml purified rWnt3aNon-canonical Wnts do not activate TOPflash
HEK293s transfectedwith indicated ligands
TOP
flash
activ
ity
Lu et al, PNAS, 2004, 101(9):3118–3123.
HEK293 cells + 200ng/ml purified rWnt3a
HEK293 cells + 200ng/ml purified rWnt3a
Gujral & MacBeath, PLoS ONE, 2010, 5(4):e10024.
WT X
Fzd’s sFRP’sCanonical Wnt’s
X
non‐canonical Wnt’sβ-catenin
sFRP’s
non-canonical Wnt’s
Fzd’s
β-catenin
X
Canonical Wnt’s
XsFRP RNAi
Wnt4/5B/11 RNAisFRP’s
X
Fzd’s Canonical Wnt’s
X
non‐canonical Wnt’sβ-catenin
Model prediction: Large under- and over-expression of bothnon-canonical WNT’s and sFRP’s can reduce TOPflash activity
0.2 0.4 0.6 0.8 1.0 1.2 0.2 0.4 0.6 0.8 1.0 1.2Ste
ady-
stat
e le
vel o
f ava
ilabl
e Fz
dre
cept
ors
(A.U
.)RNAi
RNAi
“WT total non-canonical WNT level”“WT SFRP level”
OE
OE
“WT total non-canonical WNT level”“WT SFRP level”
O
O
O
O
O
O
Non-canonical WNT’s, amount (A.U.) sFRP’s , amount (A.U.)
RNAi RNAi OE OE
– Wnt + Wnt – Wnt + Wnt
Ub Core UBE2M, CUL1, DUSP18, βTRCP
SKP1A ↑lower β-cat degradation
↑ ↑
APC ↑AXIN 1/2 ↑ ↑ M ↑ M ↓ M
SIAH1 ↑ M (degrades β-cat)
SMURF1/2 ↓ M (degrades Axin) ↓ (activates PCP)
TCF/LEF1 ↓ ↑ ↑LRPs/FZs ↓
Wnt4/5B/11 ↓ M
sFRP2 ↓ M
WIF1 ↑FRAT1/GNAi1 ↓ ↓ ↑ ↑
PP1γ ↓ (as Axin OE)
DVL1/2/3 ↑ X (see idea) Miram’s ↓ ↑ Miram’s ↑ ↑ Miram’s ↑NKD1 ↓ M ↓ M
GSK3α/β ↓ X GSK3β titration? α↓ ↓β M Miram’s ↑ ↓ M Miram’s ↓CSNK2A1/2 ↑ (β-cat degradation) ↑ (β-cat degradation)
CSNK1ε ↓ (signalosome) ↑ (signalosome) ↑ (signalosome)
CSNK1δ ↑ (in DC) ↓ (in signalosome) ↑ (in signalosome) ↑ (in signalosome)
CTNNB1 ↓ ↓ ↑ ↑
proteasomal degradation of Axin and APC
CSK2 is constitutively active
WT DVL Nkd1
Axin1+Axin2(+ etc)
Dvl
signalosomeWnt Destruction Complex
β-catenin
NKD1AXIN2
Nkd1
Axin1+Axin2
signalosome
Dvl
Destruction Complex
β-catenin
NKD1AXIN2
Wnt
Nkd1
Axin1+Axin2
signalosome
Dvl
Destruction Complex
β-catenin
NKD1AXIN2
Wnt
OE RNAi ?
Interesting hypotheses to test experimentally:
1. Is there a Wnt response threshold in single cells?
2. Do OE and RNAi of sFRPs and non-canonical Wnts both reduce TOPflash activity?
3. Do DVL RNAi and OE both reduce TOPflash activity?
4. Does Nkd1 bind DVL more strongly than Axin?
5. Does over-expressed DVL recruit Axin out of the destruction complex?
Only mechanistically correct models can extrapolate reliability
“The man who has fed the chicken every day throughout its life at last wrings its neck instead, showing that more refined views as to the uniformity of nature would have been useful to the chicken.”
– Bertrand Russell in The Problems of Philosophy, 1912, Chapter 6. .
The Yin-Yang of genetics:
The “a few bad apples” scenario:
A small number of distinct causes underlie the vast majority of cases of any disease.
Once we know the perpetrator(s), the same intervention(s) can be applied to all cases.
The Anna Karenina scenario:(Every unhappy family is unhappy in its own way.)
Dysregulation is the result of different combinations of causes in each individual.
Interventions must be individualized.
( )
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( )
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( )( )
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( ) ( )
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( ) ( )
Wnt pathway gene expression in 108
normal and tumor prostate samples from 54 patients.
Wissman et al, J Pathology , 2003, (201): 204–212.
DNA sequence variations affecting cellular signaling genes in two individuals
overlapWatson total Venter total (non-synonymous + frameshift)
VEGF Wnt
18 32 15 29 26 27
Watson Venter Watson Venter(883) (1273) total entries
in dbSNP
Notch Phosphatidylinositol TCR
17 3 16 21 34 27 27 22 20
Watson Venter Watson Venter Watson Venter(1250) (1010) (1141)
Ca
49 53 46
Watson Venter(2740)
Hh
7 17 15
Watson Venter(501)
BCR
26 29 23
Watson Venter(805)
ErbB
17 15 12
Watson Venter(1122)
JakStat
55 22 25
Watson Venter(1468)
MAPK
63 66 61
Watson Venter(3792)
mTOR
11 9 9
Watson Venter(1068)
TLR
23 20 19
Watson Venter(1033)
TGFβ
7 20 8
Watson Venter(720)
(Stephen Quake’s genome)
Thanks to my collaborators:
Laboratories of Randy Moon (HHMI) and Andy Chien at UW
Jason Berndt, Travis Biechele, Richard James, et al.
Thanks to my collaborators:
Laboratories of Randy Moon (HHMI) and Andy Chien at UW
Jason Berndt, Travis Biechele, Richard James, et al.