survival and death pathways in cancercancermeetings.org/thinktank/presentations/mak.pdf · dj-1...
TRANSCRIPT
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Think Tank on Molecular Targets:Think Tank on Molecular Targets:
Survival and Death Pathways in CancerSurvival and Death Pathways in Cancer
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Oncogenes Induce Cell Proliferation & Cell Death
ONCOGENESMitogens
Proliferation
Apoptosis
Adapted from G Evan
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Survival Signals Block Oncogene-Induced Cell Death
ONCOGENESMitogens
Survival Signals
Proliferation
Apoptosis
Drugtargets
?
Adapted from G Evan, Strasser, Adams, Cory et al
(PI3’K, Bcl-2, NF-κB)
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FASDR3 DR4
DR5
TRADD
FADD
DNA damage
Caspase-8
cFLIP
MitochondriaApaf-1
Caspase-9
Caspase-3
Bcl-2, Bcl-XL
RIP
TRAF2
cIAPs
IL1-R CD40OPGL-R
TRAF6
IRAKs
Receptor PTK
PI3K
PKB (Akt)
GSK3
p53
Bad
TNF-R2
PTEN
NEMO
Bid
ATM
Brca1/2
ARF
E1A, myc, ras
TNF-R1
DR6
FKHR
FAS-L
TLR
CD30
PIDDp53AIP1
NoxaT2K
TANK
MyD88
TCR
IKKs
NF-kB
Vav
PKCθ
aPKC
Bcl-10
Smac
NIK
p62
??
Chk2
Bax
Nod-1
Nod-1RICK
RICK
Apoptosis and Survival SignalsApoptosis
Malt1
Bimp
TelomereTelomerep53-bk
HBxHBx
P21
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Regulation of Cell Survival
1) Hodgkin’s; 2) MALT Lymphomas
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IL13 H/RSCell IL
-13
Rα
1
IL-4
Rα
IL13HodgkinHodgkin’’ssLymphomaLymphoma
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HDLM2-derived tumors in NOD/SCID miceCD30
P-STAT6
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Individual Data Points HDLM2 NOD/SCID
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48 hrs 72 hrs 48 hrs 72 hrs
TNF-
α S
ecre
tion
(pg/
ml)
0
100
200
300
400
Untreated controlAnti-isotype control Anti-IL13
HDLM2 L1236
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Isoty
pe co
ntro
l
a-IL-
13IL4
Ry
Probe:
Treatment:
NF-kB
1 2 3 4 5 76
Untre
ated
a-IL-
13 / I
L-4R
a-IL-
13 +
IL-4
a-IL-
13 +
IL-13
NFkBNFkB Activation in Hodgkin Cells is Dependent on ILActivation in Hodgkin Cells is Dependent on IL--1313
NF-kB survival
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Positive Feedback Loops in HDNon-EBV EBV
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IKKα IKKβNEMO
IκBα
NF-κB
TCR BCR TNF IL-1LPS
PKC?
TRADD
RIP
MyD88
TRAF6
IRAK
Ubiquitination and Degradation
Nuclear Translocation
BCL10
PMA
+P
PKCθ
?
P
MALT1
MALT Lymphomas
Ruland et al Cell, 2002Immunity 2004
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OverexpressionOverexpression of Mutant Bcl10 of Mutant Bcl10 induces constitutively active induces constitutively active NFNFκκBB
CARD
1 233
N C CARD
1
N
NFNFκκBB
NFNFκκBB(SURVIVAL)(SURVIVAL)
Valcade
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PARK7Cytochrome c
RhoC
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PARK7 regulates PTEN functionsPARK7 regulates PTEN functions
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PTEN Regulates Cell Size, Cell Death, ProliferationPTEN Regulates Cell Size, Cell Death, Proliferation
RTKRTK
PKBPKB
PTENPTENGrowth Growth FactorFactor
PIP3PIP3PP
PP
PPPIP2PIP2PP
PP
PI3PI3’’KK
Cell DeathCell Death
Glucose Glucose metabolismmetabolism Hyper
proliferation
PDKPDK
S6KS6K
TranslationTranslation
TORTOR
TscTsc
BackmanBackman et al., Nat. Gen. 29:396 (2001)et al., Nat. Gen. 29:396 (2001)
StambolicStambolic et al., Cell 95:29 (1998)et al., Cell 95:29 (1998)MaehamaMaehama & Dixon, JBC 273:13375 (1998)& Dixon, JBC 273:13375 (1998)
PP PP--S473S473
FKHRLFKHRL
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PTEN Is a Tumor-Suppressor Gene With Mutations in
• >70% of Glioblastomas• >40% of Endometrial carcinomas• >30% of Prostate carcinomas• >25% of Melanomas• >20% of Small-cell lung cancers
< 5% of Breast carcinomas, NSCLC, Leukemias, lymphomas, etc.
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Breast CarcinomaBreast CarcinomaPKB P-PKB
Normal Normal
TumorTumor TumorTumorYYYY
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Regulation of PTEN FunctionsRegulation of PTEN Functions
1) Mutations
2) Phosphorylation
3) Methylation
4) Crosstalk with p53
5) Crosstalk with ras/raf/ERK
6) Others
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Human DJ-1 overexpressionRescues PTEN Phenotype
ey-Gal4-UAS-PTEN/CyO
UASey-Gal4/+
ey-Gal4-UAS-PTEN/UAS-DJ1
Kim et al., Can Cell 2005
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What is DJ-1?
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DJ-1 History• Cloned as an NIH3T3 transforming gene
• Part of Pfp1/ThiJ/DJ1 superfamily– All have conserved Cys-His, DJ-1 Cys106
• Upregulated by growth factors, drugs (Taxol, MEK inhibitors)
• Identified as PARK7 – Causative agent for autosomal-recessive
early-onset Parkinsonism
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Two Views: DJ-1 Dimer
X
X
Cys106Cys106
• Obligate homodimer• 189 aa, 19.8 kDa• Helix-strand-helix
sandwich structure
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DJ-1 Induces Higher Resistance to Cell Death
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Knockdown of DJ-1 Decreases pPKB/akt in PTEN-Positive Cells
Kim et al Can Cell 2005
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Elevated Expression in Lung Cancer
P80 P2P18 P19 P38P13
5P53 P31P11
7P74P22
4P68 P4P83 P91P13
3P15
9P18
1P88P15
2P20 P47 P60
0
1
2
3
4
5
6
7 NormalTumor
Rela
tive
mRN
A e
xpre
ssio
n le
vels
Cases comparing tumor to paired non-neoplastic lung
Adenocarcinoma Squamous cell carcinomaTsao, M. 2004
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High Levels of DJ-1 Increase Risk ofCumulative Incidence for Relapse in Lung Ca
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DJ-1 expression in lymphoma cell lines
0.0
0.1
0.2
0.3
0.4
0.5
0.6
DJ1 Bcl2 cmyc cyclinD1
Rel
ativ
e ex
pres
sion
ly3 ly18 OCI/Bkt1 Daudi Raji
Ly3 Ly18 Daudi Raji
DJ1 expressionRTQ-PCR
M Minden, PMH, Toronto
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SurvivalSurvivalCell DeathCell DeathCancerCancerParkinsonParkinson
DJDJ--1 Mediates Homeostasis1 Mediates Homeostasis
MPTP MPTP ––TH+ NEURONSTH+ NEURONS
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Various apoptotic stimuli
BaxBadBak
Death receptorDeath receptor
∆Ψ↓∆Ψ↓
CytochromeCytochrome cc
ApafApaf--11
CaspaseCaspase--99
CCaassppaassee--33
BBiidd
ttBBiidd
BclBcl--22BclBcl--xLxL
DIABLODIABLO((SmacSmac))
IAPIAP
AAppooppttoossiiss
FADDFADD
CaspaseCaspase--88
ActivatedActivatedCaspaseCaspase 88
LigandLigand
XExtrinsicExtrinsic IntrinsicIntrinsic
ExtrinsicExtrinsic & & IntrinsicIntrinsic PathwaysPathways
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MitochondriaMitochondria--Dependent ApoptosisDependent Apoptosis
mitochondria
Stimuli
Casp9
Apaf1Cyt c
Casp3
Apoptosis
Bcl-2
K72AK72A
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Cyt c-mediated apoptosis plays a critical rolein brain development
Hao et al Cell, 2005
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Cyt c KA Resistant to Apoptosis
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Development of lymphadenopathy and splenomegaly in KA/KA Mice
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Various apoptotic stimuli
BaxBadBak
Death receptorDeath receptor
∆Ψ↓∆Ψ↓
CytochromeCytochrome cc
ApafApaf--11
CaspaseCaspase--99
CCaassppaassee--33
BBiidd
ttBBiidd
BclBcl--22BclBcl--xLxL
DIABLODIABLO((SmacSmac))
IAPIAP
AAppooppttoossiiss
FADDFADD
CaspaseCaspase--88
ActivatedActivatedCaspaseCaspase 88
LigandLigand
XExtrinsicExtrinsic IntrinsicIntrinsic
ExtrinsicExtrinsic & & IntrinsicIntrinsic PathwaysPathways
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Differential susceptibility to cell death in cyt c K72A and apaf1 thymocytes for Dex, Etoposide, and γ-ray
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Normal caspase-3 cleavage in KA/KA thymocytes
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Casp9
Apaf1Cyt c
Casp3
Apoptosis
BaxBakBaxBak
Cytochrome c-independent caspase activation pathway in Cyt c KA/KA thymocytes
MEFs
Casp9
Cyt c
Casp3
Apoptosis
BaxBakBaxBakThymocytes
Apaf1
XFasFas
Casp8
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In Vivo Function Of RhoC
•Development•Cancer and Metastasis
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Rho GTPase Family Members
• Rac• Cdc42• Rnd• Rho (A,B,C)
There are at least 20 distinct Rho Family members divided into subfamilies:
• Rho A, B, and C share over 85% homology. • Activation of Rho is regulated by GAP, GEF, and GDI.
MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYIA DIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPDSLENIPEKWTPQVKHFCPNVPIILVGNKKDLRQDEHTRRELAKMKQEPVRSEEGRDMANRISAFGYLECSAKTKEGVREVFEMATRAGLQVRKNKRRRGCPIL
RhoC Protein
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RhoGEF
Rho Gap
Rho GDI
GTP
Rho
Effector
RhoGDI
Rho GDI
RhoGEF
Rho
Rho
GTP
GDP
GDP
Pi
UbUb
Regulation of Rho-Family GTPases
Possible Rho effectors:
mDia(Actin organization)Rock
RhophilinCitron (Cytokinesis)PI-4-P5k (PIP2 level
shape-mobility-CC interact-G1 phase, c-fos, c-jun
Bishop and Hall ‘00
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Rho Family Members in Metastasis
RhoA can transform and enhance invasiveness in certain cells (Yoshioka et al., 1999)
Overexpression of RhoC increases angiogenicfactors in breast cells in vitro (van Golen et al., 2000)
Overexpression of RhoC elevates melanoma cells; exits blood to colonize lungs (Clark et al., 2000)
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RhoC is not essential in T- or B-cell development and activation
+/+
-/-
-/-+/+
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RhoC is not essential in T- or B-Cell apoptosis or migration
+/--/-
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RhoC: required for stress fiber formation in MEFs
+FCS
-FCS 48hr
No differences in apoptosis (UV, γ); transformation (E1A/Ras)
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Role of RhoC in Tumor Formation and Metastasis
1) Tumor formation in RhoC-/- mice
2) Metastasis in RhoC-/- micea) motilityb) angiogenesisc) cell death
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RhoC: not essential for primary tumor formation & death(PyV-mT)
H&E TunelNo Differences:
No. of tumors
Size of tumors
Structure
Proliferation-Ki67
Angiogenesis-CD31, Factor VIII
Tunel
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RhoC is essential for metastasis
H&E
+/-170+30U
-/-12+5U
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RhoC is Involved in Motility and Invasiveness
Transwell: in response to SDF-1
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RhoC Affects Apoptosis in Lung Metastases
+/-
-/-
Cleaved caspase 3
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Role of RhoC in Tumor Formation and Metastasis
1) Tumor formation in RhoC-/- mice N
2) Metastasis in RhoC-/- micea) motility Impairedb) angiogenesis “normal”?c) cell death enhanced
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Future DirectionsFuture Directions
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Survival and Death Pathways Are Ideal Targets
ONCOGENE
Mitogens
Survival Signals
Proliferation
Apoptosis
DrugTargets
(PI3’K, Bcl-2, NF-κB)
Metastasis
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Imatinib MesylateImatinib Mesylate ((GleevecGleevec): ): An Inhibitor of Cell Survival An Inhibitor of Cell Survival
proliferation survival
Ras-raf-MAPK PI3K-PKB/Akt
PP P
P
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Hodgkins Hodgkins U U KappKapp R R GascoyneGascoyneB B Skinnider Skinnider B PatersonB Paterson
V V StambolicStambolicM PetersM PetersR KimR Kim
PTEN/DJPTEN/DJ--11
Bcl10/Malt1 Bcl10/Malt1 J J RulandRuland P P Ohashi Ohashi G DuncanG Duncan
M TsaoF LiuM Minden
RhoC RhoC A A HakemHakem R R KhohkaKhohkaCytCyt c c Z Z HaoHao X WangX Wang