Think Tank on Molecular Targets:Think Tank on Molecular Targets:

Survival and Death Pathways in CancerSurvival and Death Pathways in Cancer

Oncogenes Induce Cell Proliferation & Cell Death

ONCOGENESMitogens

Proliferation

Apoptosis

Adapted from G Evan

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)

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

Regulation of Cell Survival

1) Hodgkin’s; 2) MALT Lymphomas

IL13 H/RSCell IL

-13

Rα

1

IL-4

Rα

IL13HodgkinHodgkin’’ssLymphomaLymphoma

HDLM2-derived tumors in NOD/SCID miceCD30

P-STAT6

Individual Data Points HDLM2 NOD/SCID

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

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

Positive Feedback Loops in HDNon-EBV EBV

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

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

PARK7Cytochrome c

RhoC

PARK7 regulates PTEN functionsPARK7 regulates PTEN functions

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

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.

Breast CarcinomaBreast CarcinomaPKB P-PKB

Normal Normal

TumorTumor TumorTumorYYYY

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

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

What is DJ-1?

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

Two Views: DJ-1 Dimer

X

X

Cys106Cys106

• Obligate homodimer• 189 aa, 19.8 kDa• Helix-strand-helix

sandwich structure

DJ-1 Induces Higher Resistance to Cell Death

Knockdown of DJ-1 Decreases pPKB/akt in PTEN-Positive Cells

Kim et al Can Cell 2005

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

High Levels of DJ-1 Increase Risk ofCumulative Incidence for Relapse in Lung Ca

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

SurvivalSurvivalCell DeathCell DeathCancerCancerParkinsonParkinson

DJDJ--1 Mediates Homeostasis1 Mediates Homeostasis

MPTP MPTP ––TH+ NEURONSTH+ NEURONS

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

MitochondriaMitochondria--Dependent ApoptosisDependent Apoptosis

mitochondria

Stimuli

Casp9

Apaf1Cyt c

Casp3

Apoptosis

Bcl-2

K72AK72A

Cyt c-mediated apoptosis plays a critical rolein brain development

Hao et al Cell, 2005

Cyt c KA Resistant to Apoptosis

Development of lymphadenopathy and splenomegaly in KA/KA Mice

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

Differential susceptibility to cell death in cyt c K72A and apaf1 thymocytes for Dex, Etoposide, and γ-ray

Normal caspase-3 cleavage in KA/KA thymocytes

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

In Vivo Function Of RhoC

•Development•Cancer and Metastasis

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

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

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)

RhoC is not essential in T- or B-cell development and activation

+/+

-/-

-/-+/+

RhoC is not essential in T- or B-Cell apoptosis or migration

+/--/-

RhoC: required for stress fiber formation in MEFs

+FCS

-FCS 48hr

No differences in apoptosis (UV, γ); transformation (E1A/Ras)

Role of RhoC in Tumor Formation and Metastasis

1) Tumor formation in RhoC-/- mice

2) Metastasis in RhoC-/- micea) motilityb) angiogenesisc) cell death

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

RhoC is essential for metastasis

H&E

+/-170+30U

-/-12+5U

RhoC is Involved in Motility and Invasiveness

Transwell: in response to SDF-1

RhoC Affects Apoptosis in Lung Metastases

+/-

-/-

Cleaved caspase 3

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

Future DirectionsFuture Directions

Survival and Death Pathways Are Ideal Targets

ONCOGENE

Mitogens

Survival Signals

Proliferation

Apoptosis

DrugTargets

(PI3’K, Bcl-2, NF-κB)

Metastasis

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

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