Think Tank on Molecular Targets: Survival and Death Pathways in Cancer

Think Tank on Molecular Targets: Survival and Death Pathways in Cancer

Oncogenes Induce Cell Proliferation & Cell Death Proliferation Mitogens ONCOGENES Apoptosis Adapted from G Evan

Survival Signals Block Oncogene-Induced Cell Death Proliferation Mitogens ONCOGENES? Apoptosis Drug targets Survival Signals (PI3 K, Bcl-2, NF-κB) Adapted from G Evan, Strasser, Adams, Cory et al

TCR Vav PKCθ RICK apkc p62 Bimp Bcl-10 Malt1 IL1-R TLR MyD88 IRAKs TRAF6 Nod-1 NIK CD30 CD40 OPGL-R?? TRAF2 TANK T2K NEMO IKKs NF-kB TNF-R2 RIP ciaps TNF-R1 DR3 DR6 TRADD cflip RICK Smac Caspase-3 FAS DR4 DR5 FADD Caspase-8 Apaf-1 Caspase-9 Telomere Bid Nod-1 p53 Mitochondria Bcl-2, Bcl-X L DNA damage E1A, myc, ras ATM ARF Chk2 PIDD p53aip1 Noxa Bax HBx Brca1/2 p53-bk P21 Bad Receptor PTK PI3K PKB (Akt) FKHR FAS-L PTEN GSK3 Apoptosis Apoptosis and Survival Signals

Regulation of Cell Survival 1) Hodgkin s; 2) MALT Lymphomas

Hodgkin s Lymphoma IL13 IL13 H/RS Cell IL-13 Rα1 IL-4 Rα

HDLM2-derived tumors in NOD/SCID mice CD30 P-STAT6

Individual Data Points HDLM2 NOD/SCID

TNF- α Secretion (pg/ml) 400 300 200 100 Untreated control Anti-isotype control Anti-IL13 0 48 hrs 72 hrs 48 hrs 72 hrs HDLM2 L1236

NFkB Activation in Hodgkin Cells is Dependent on IL-13 Probe: NF-kB Untreated Isotype control a-il-13 IL4Ry a-il-13 / IL-4R a-il-13 + IL-4 a-il-13 + IL-13 Treatment: NF-kB survival 1 2 3 4 5 6 7

Non-EBV EBV Positive Feedback Loops in HD

? TCR BCR LPS TNF IL-1 PMA PKCθ BCL10 MALT1 MALT Lymphomas PKC? NEMO IKKα IKKβ IκBα NF-κB +P P TRADD MyD88 IRAK RIP TRAF6 Ubiquitination and Degradation Nuclear Translocation Ruland et al Cell, 2002 Immunity 2004

Overexpression of Mutant Bcl10 induces constitutively active NFκB 1 233 1 N CARD C N CARD NFκB Valcade NFκB (SURVIVAL)

PARK7 Cytochrome c RhoC

PARK7 regulates PTEN functions

PTEN Regulates Cell Size, Cell Death, Proliferation Growth Factor P PTEN P P RTK PIP2 P PI3 K PIP3 P PDK P PKB P-S473 S6K Glucose metabolism Stambolic et al., Cell 95:29 (1998) Maehama & Dixon, JBC 273:13375 (1998) Backman et al., Nat. Gen. 29:396 (2001) FKHRL Cell Death Tsc Hyper proliferation TOR Translation

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.

Y Y Breast Carcinoma PKB Normal Normal P-PKB Tumor Tumor

Regulation of PTEN Functions 1) Mutations 2) Phosphorylation 3) Methylation 4) Crosstalk with p53 5) Crosstalk with ras/raf/erk 6) Others

Human DJ-1 overexpression Rescues PTEN Phenotype UAS ey-gal4/+ ey-gal4-uas- PTEN/CyO 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 Cys106 X 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 Cases comparing tumor to paired non-neoplastic lung 7 Normal 6 Tumor 5 4 3 2 1 0 P80 P2 P18 P19 P38 P135 P53 P31 P117 P74 P224 P68 P4 P83 Relative mrna expression levels P91 P133 P159 P181 P88 P152 P20 P47 P60 Tsao, M. 2004 Adenocarcinoma Squamous cell carcinoma

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

DJ-1 expression in lymphoma cell lines RTQ-PCR DJ1 expression Relative expression 0.6 0.5 0.4 0.3 0.2 0.1 0.0 DJ1 Bcl2 cmyc cyclind1 Ly3 Ly18 Daudi Raji ly3 ly18 OCI/Bkt1 Daudi Raji M Minden, PMH, Toronto

DJ-1 1 Mediates Homeostasis MPTP TH+ NEURONS Cell Death Parkinson Survival Cancer

Extrinsic & Intrinsic Pathways Ligand Death receptor Various apoptotic stimuli FADD Bax Bad Bak Ψ Bcl-2 Bcl-xL Caspase-8 tbid Extrinsic Activated Caspase 8 Bid Cytochrome c Apaf-1 X Caspase-9 DIABLO (Smac) IAP Intrinsic Caspase-3 Apoptosis

Mitochondria-Dependent Apoptosis Stimuli Bcl-2 mitochondria Apaf1 Cyt c K72A Casp3 Casp9 Apoptosis

Cyt c-mediated apoptosis plays a critical role in brain development Hao et al Cell, 2005

Cyt c KA Resistant to Apoptosis

Development of lymphadenopathy and splenomegaly in KA/KA Mice

Extrinsic & Intrinsic Pathways Ligand Death receptor Various apoptotic stimuli FADD Bax Bad Bak Ψ Bcl-2 Bcl-xL Caspase-8 tbid Extrinsic Activated Caspase 8 Bid Cytochrome c Apaf-1 X Caspase-9 DIABLO (Smac) IAP Intrinsic Caspase-3 Apoptosis

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

Cytochrome c-independent caspase activation pathway in Cyt c KA/KA thymocytes MEFs BaxBak X Thymocytes BaxBak Fas Apaf1 Cyt c Apaf1 Cyt c Casp3 Casp9 Casp9 Casp3 Casp8 Apoptosis Apoptosis

In Vivo Function Of RhoC Development Cancer and Metastasis

Rho GTPase Family Members There are at least 20 distinct Rho Family members divided into subfamilies: Rac Cdc42 Rnd Rho(A,B,C) Rho A, B, and C share over 85% homology. Activation of Rho is regulated by GAP, GEF, and GDI. RhoC Protein MAAIRKKLVIVGDGACGKTCLLIVFSKDQFPEVYVPTVFENYIA DIEVDGKQVELALWDTAGQEDYDRLRPLSYPDTDVILMCFSIDSPD SLENIPEKWTPQVKHFCPNVPIILVGNKKDLRQDEHTRRELAKMKQ EPVRSEEGRDMANRISAFGYLECSAKTKEGVREVFEMATRAGL QVRKNKRRRGCPIL

Regulation of Rho-Family GTPases Possible Rho effectors: Rho GEF Ub Ub GTP GDP Rho GEF Rho GDI Rho GDP GTP Rho Rho Rho GDI Effector mdia(actin organization) Rock Rhophilin Citron (Cytokinesis) PI-4-P5k (PIP2 level shape-mobility-cc interact- G1 phase, c-fos, c-jun Rho GDI Rho Gap Pi 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 angiogenic factors 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-/- mice a) motility b) angiogenesis c) cell death

RhoC: not essential for primary tumor formation & death (PyV-mT) H&E No Differences: No. of tumors Size of tumors Structure Proliferation-Ki67 Angiogenesis- CD31, Factor VIII Tunel 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-/- mice a) motility Impaired b) angiogenesis normal? c) cell death enhanced

Future Directions

Survival and Death Pathways Are Ideal Targets Mitogens Proliferation ONCOGENE Metastasis Apoptosis Survival Signals (PI3 K, Bcl-2, NF-κB) Drug Targets

Imatinib Mesylate (Gleevec): An Inhibitor of Cell Survival P P Ras-raf-MAPK P P PI3K-PKB/Akt proliferation survival

PTEN/DJ-1 Hodgkins Bcl10/Malt1 J Ruland G Duncan RhoC A Hakem Cyt c Z Hao V Stambolic M Tsao M Peters F Liu R Kim M Minden U Kapp R Gascoyne B Skinnider B Paterson P Ohashi R Khohka X Wang