A Translational Investigation of Metastasis Ning Zhang Tianjin - - PowerPoint PPT Presentation

A Translational Investigation

• f Metastasis

Ning Zhang Tianjin Medical University

Metastasis of Cancer Cells

What is Chemotaxis?

• Leukocyte trafficking
• Angiogenesis
• Wound healing
• Brain Development
• HIV infection

αi"

β" β" γ" γ" chemokine!

Correlation between Chemotaxis and Cancer Metastasis

CXCR4 mediates chemotaxis of human breast cancer cells

Zlotnik Nature 2001

medium EGF CXCL12 5 10 15 Chemotaxis Index

ng/ml

1 10 100 100 1000 1000

EGF is a more potent chemoattractant than CXCL12 than CXCL12

MDA-MB-231 cells

chemokine Cells membrane

Chemotaxis

GPCR EGFR

P P P P

PLCγ" γ"

?"

PLCβΙΙ" ΙΙ" Gi protein

PKC Family

βI, βII, γ" βI, βII, γ"

• Novel, DAG-dependent, such as δ, ε, θ, η.
• Atypical, doesn’t require either Ca2+ or DAG,

such as ζ and λ.

EGF Ly294002

• PKCζ"

PKCα"

EGF induces PKCζ translocation

Activated PKCζ regulates directional cell migration and polarity

PKCζ Pseudosubstrate inhibits EGF- induced chemotaxis

MCF-7 T47D MDA-231

• M

e d i u m Myristoylated 50 µM Non-Myristoylated 50 µM ng/ml ng/ml ng/ml ng/ml EGF (ng/ml)

MCF-7 T47D MDA-231 MCF-7 T47D MDA-231

Pretreatments Chemotaxis Index

Cancer Research 2005

Normal Carcinoma Lymph Node

Expression of PKCζ Correlates with Lymph Node Metastasis

Rictor Interacts with PKCζ

PKCζ colocalizes with Rictor along plasma membrane under EGF stimulation

PKCζ colocalizes with Rictor at the leading edge of migratory cells

Knockdown of Rictor by siRNA Decreases EGF Induced Chemotaxis

MDA-MB-231 Scr clone 4 clone 11 clone 15

20 40 60 80

1 10 100 EGF(ng/ml)

***

Cell number/HPF

Knockdown of Rictor Impairs EGF Induced PKCζ Membrane Translocation

Rictor and Raptor Define Two Distinct mTOR Containing Complexes

Kim DH, Cell, 2002

Rictor still co-immunoprecipitated with PKCζ in siSIN1 cells

EGF-induced actin polymerization was impaired in siRictor cells

6 weeks after implantation Scr: 6 weeks siRictor: 9 weeks

Knockdown of Rictor Inhibited Spontaneous Metastasis of MDA-MB-231 Cells to SCID Mouse Lung

Cancer Research 2010

Expression of Rictor is Linked with Lymph Node Metastasis of Breast Cancer tissues

positive negative total normal 3 36 39 cancer 25 14 39 total 28 50 78

p=0.000

rictor Parameters/Markers Total Positive % p Menopausal 0.261 Pre-menopausal 21 12 57.1 Post-menopausal 18 13 72.2 Tumor Size <2 cm 7 5 71.4 .507 >2 cm 32 20 62.5 Lymph Node Status Negative 13 1 0.07 0.000 Positive 26 24 92.3 Histological Grade G1 13 9 69.2 0.780 G2 22 14 63.6 G3 4 2 50.0 ER Status Negative 16 7 43.8 0.031 Positive 23 23 100 PR Status Negative 18 10 55.6 0.243 Positive 21 15 71.4 HER2/neu Protein Negative 29 16 55.2 0.049 Positive 10 9 90.0

Rictor is Expressed in Lung Cancer Tissues

Expression of Rictor correlates with NSCLC lymph node metastasis and poor prognosis

Rictor was Expressed in Renal Cancer tissues

Expression of Rictor in Renal Cancer Correlates with Poor Prognosis

Chemotaxis

GPCR EGFR

P P P P

PLCγ" γ" PI3Kα/δ?"

PKCζ" ζ"

PLCβΙΙ" ΙΙ" PI3Kγ/δ" δ" Gi protein LIMK1/Cofilin Integrinβ1 (Actin Polymerization) (Adhesion)

Rictor"

Chemotaxis GPCR EGFR

P P P P

PLCγ

PKCζ"

Gi protein PTEN PDK1 Akt2 PLCβ" β"

PLA2 ??

Rictor

Substrates ??

Current Working Model

Cancer Research 2005 Mol Membrane Bio 2007 Cell Signal. 2007 Cell Signal. 2008 Lung Cancer 2008 International J Cancer 2009 Mol Cancer Research 2009 J Proteome research 2009 Cancer Research 2010 J Clinical Invest. 2010

Chemotaxis

GPCR EGFR

P P P P

PI3Kα/δ?" PKCζ" ζ" PI3Kγ/δ" δ" Gi protein AKT2 PDK1

Compound Library

Biochemical siRNA Cell-Based Animal Model Clinical Novel Drug

So What?

Transform of drug 5-8 plus 572625( control)

10-7 10-6 10-5 10-4 10-3 20 40 60 80 100 120

9021878 7750407 5119533 5734879 572625 M % Activity

Screening for PKCζ inhibitors

• A Cell Based Screening for the

Inhibitors of PKCζ"

EGF

• +

+ +

Akt inhibitor LY294002

Anal Biochem 2007

Invest New Drugs 2009 Cancer Letters 2010

Acknowledgements

Zhang Fei Guo Hua Zhang Baogang Zhang Xiaofang Tian Gang Liu Yan Sun Ronghua Liu Ying Wan Wuzhou Wang Jingna

Metastasis is the Major Cause of Morbidity

Rictor β-actin

Knockdown of Rictor by siRNA Decreases EGF Induced Chemotaxis

T47D Scr siRictor siPKCz 10 20 30 40 50

1 10 100 EGF(ng/ml)

**

Cell number/HPF

Knockdown of Rictor by siRNA Decreases EGF Induced Chemotaxis

5 15 30 30 60 90 120 150

Scr EGF (-) Scr EGF (+) siRictor EGF (-) siRictor EGF (+) EGF(10 ng/ml)

Time(min) Cell number/HPF

Cell adhesion was Impaired in siRictor cells

EGF still induces Rictor membrane translocation in siPKCζ cells

EGF (-) EGF (+) 20 40 60 80 100

MDA/Scr MDA/siPKCζ

Cells with Rictor translocation(%)

EGF induced PKCz phosphorylation was impaired in siRictor cells

Scr siRictor

2 4 6 8

30'' 1' 2' 5'

Time

*

PKCζ phosphorylation

WCL NC 0’ 5’ 30’ IP: Akt2 PKCζ Akt2 NC 0’ 5’ 30’ IP: PKC ζ Akt2 WCL PKCζ 0’ 5’ 30’ IP: Akt1 WCL NC Akt1 Akt1 0’ 5’ 30’ WCL NC PKCζ PKCζ IP: PKCζ

EGF Induces Co-IP of Akt2 and PKCζ"

Akt2 Plays a Critical Role in Metastasis

EGF (ng/ml) 1 10 100 Chemotaxis Index 2 4 6 8 10 12 14 Control C44 C97 C102

• Akt2

Akt1 Control C44 C98 C97 C102 p-cofilin p-LIMK cofilin 30’’ 1’ 2’ 5’ 30’‘ 1’ 2’ 5’ Contro l siAkt2/MDA

PDK1 is Required for Metastasis

An Optimal Level of PTEN is required for Chemotaxis

Chemtoaxis Index 1 10 100 EGF (ng/ml) 1000 MDA-MB-231 V5-His-PTEN/ MDA-MB-231 5 10 15

• PTEN

V5-His-PTEN 1 2 Chemtoaxis Index 1 10 100 EGF (ng/ ml) 1000 MDA Control Clone 6 Clone 100 Clone 74 4 8 12

• PTEN

GAPDH Control M DA Clone 6 Clone 74 Clone100

PDK1 Control siPTEN/MDA

• JCI 2010

TXNL2 ! ROS ! GSH ! NFkB ! metastasis

What’s Next?

Co-immunoprecipitation MDA-MB-231 cell HEK293 cell

P32

scr sip32 Golgi(Giantin)

scr sip32

B23

SCR siB23 SCR SiB 20min 6h

cPLA2a

Treatments with Gd@C82(OH)22 Inhibit hepatoma growth in a mouse model

Nano Lett Vol 5, pg 2050 Gd@C82(OH)22 inhibits tumor growth in a breast tumor model. 0.28 mg/kg 1.20mg/kg

Gd@C82(OH)22 induced iDC maturation and TH-1 response.

ACS Nano 2010

0.00 2.00 4.00 6.00 8.00 10.00 1 10 100 1000 Chemotaxis Index 0 uM 1 uM 10 uM

[EGF] (ng/ml)

Treatments Metastasis Rate Saline q.d. ×20 day 66.7% C60(C(COOH)2)2 (0.4 mg/kg, n = 10) 34.2% C60(OH)20 (0.4 mg/kg, n = 10) 38.0% Gd@C82(OH)22 0.35mg/kg q.d. ×20 day 4.3 %

Treatments with Gd@C82(OH)22 cancer cell chemotaxis and metastasis

Summary

• 1. Treatment with Gd@C82(OH)22 inhibits tumor growth

without detectable toxicity.

• 2. Gd@C82(OH)22 doesn’t show cytotoxicity.
• 3. Gd@C82(OH)22 inhibits blood supply to tumor tissues.
• 4. Gd@C82(OH)22 induced tumor immunity.
• 5. Gd@C82(OH)22 inhibits cancer cell chemotaxis

Blood supply Tumor immunity Metastasis