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

A Translational Investigation of Metastasis Ning Zhang Tianjin Medical University

Metastasis of Cancer Cells

What is Chemotaxis? chemokine ! • Leukocyte trafficking α i " γ" γ" • Angiogenesis β" β" • Wound healing • Brain Development • HIV infection

Correlation between Chemotaxis and Cancer Metastasis CXCR4 mediates chemotaxis of human breast cancer cells Zlotnik Nature 2001

EGF is a more potent chemoattractant than CXCL12 than CXCL12 15 ng/ml Cells 1 10 Chemotaxis Index 10 100 100 membrane 1000 1000 chemokine 5 0 medium EGF CXCL12 MDA-MB-231 cells

EGFR GPCR P P P P Gi protein PLC βΙΙ" PLC γ" ΙΙ" γ" ? " Chemotaxis

PKC Family β I, β II, γ" β I, β II, γ" • Novel, DAG-dependent, such as δ , ε , θ , η . • Atypical, doesn’t require either Ca 2+ or DAG, such as ζ and λ .

EGF induces PKC ζ translocation ��� �� �� EGF ��� ��� �� Ly294002 PKC ζ" PKC α"

Activated PKC ζ regulates directional cell migration and polarity

PKC ζ Pseudosubstrate inhibits EGF- induced chemotaxis ��� EGF (ng/ml) �� ng/ml ��� Chemotaxis Index �� ng/ml ��� ng/ml ��� ���� ng/ml ��� �� �� MDA-231 MCF-7 T47D MDA-231 MCF-7 T47D MDA-231 MCF-7 T47D M e d i u m Non-Myristoylated Myristoylated 50 µ M 50 µ M Pretreatments Cancer Research 2005

Expression of PKC ζ Correlates with Lymph Node Metastasis Normal Carcinoma Lymph Node

Rictor Interacts with PKC ζ

PKC ζ colocalizes with Rictor along plasma membrane under EGF stimulation

PKC ζ colocalizes with Rictor at the leading edge of migratory cells

Knockdown of Rictor by siRNA Decreases EGF Induced Chemotaxis 80 EGF(ng/ml) 0 Cell number/HPF 1 10 60 100 *** 40 20 0 MDA-MB-231 Scr clone 4 clone 11 clone 15

Knockdown of Rictor Impairs EGF Induced PKC ζ Membrane Translocation

Rictor and Raptor Define Two Distinct mTOR Containing Complexes Kim DH, Cell, 2002

Rictor still co-immunoprecipitated with PKC ζ in siSIN1 cells

EGF-induced actin polymerization was impaired in siRictor cells

Knockdown of Rictor Inhibited Spontaneous Metastasis of MDA-MB-231 Cells to SCID Mouse Lung Scr: 6 weeks 6 weeks after implantation siRictor: 9 weeks 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 Pre-menopausal 21 12 57.1 0.261 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 G2 22 14 63.6 0.780 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

EGFR GPCR P P P P Gi protein PI3K α / δ ? " PI3K γ / δ" δ" PLC βΙΙ" PLC γ" ΙΙ" γ" Rictor " PKC ζ" ζ" LIMK1/Cofilin Integrin β 1 (Actin Polymerization) (Adhesion) Chemotaxis

Current Working Model EGFR GPCR P P P P Gi protein PTEN PLC γ PLC β" β" Cancer Research 2005 PDK1 Rictor PLA2 ?? Mol Membrane Bio 2007 Akt2 Cell Signal. 2007 Cell Signal. 2008 Lung Cancer 2008 International J Cancer 2009 PKC ζ" Mol Cancer Research 2009 J Proteome research 2009 Substrates ?? Cancer Research 2010 J Clinical Invest. 2010 Chemotaxis

So What? GPCR Compound Library � siRNA EGFR Biochemical P P P P Gi protein Cell-Based PI3K γ / δ" PI3K α / δ ? " δ" Animal Model PDK1 AKT2 Clinical PKC ζ" ζ" Novel Drug Chemotaxis

Screening for PKC ζ inhibitors Transform of drug 5-8 plus 572625( control) 9021878 120 7750407 5119533 100 5734879 572625 80 % Activity 60 40 20 0 10 -7 10 -6 10 -5 10 -4 10 -3 M

A Cell Based Screening for the Inhibitors of PKC ζ" 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

Knockdown of Rictor by siRNA Decreases EGF Induced Chemotaxis Rictor β -actin

Knockdown of Rictor by siRNA Decreases EGF Induced Chemotaxis EGF(ng/ml) 50 0 Cell number/HPF 1 40 10 100 ** 30 20 10 0 T47D Scr siRictor siPKCz

Cell adhesion was Impaired in siRictor cells 150 EGF(10 ng/ml) Scr EGF (-) Scr EGF (+) 120 Cell number/HPF siRictor EGF (-) siRictor EGF (+) 90 60 30 0 5 15 30 Time(min)

EGF still induces Rictor membrane translocation in siPKC ζ cells 100 MDA/Scr MDA/siPKC ζ 80 Cells with Rictor translocation(%) 60 40 20 0 EGF (-) EGF (+)

EGF induced PKCz phosphorylation was impaired in siRictor cells Time PKC ζ phosphorylation * 8 0 30'' 1' 6 2' 5' 4 2 0 Scr siRictor

EGF Induces Co-IP of Akt2 and PKC ζ" IP: Akt2 IP: PKC ζ WCL 5’ 30’ WCL NC 0’ 5’ 30’ NC 0’ Akt2 PKC ζ PKC ζ Akt2 IP: PKC ζ IP: Akt1 WCL 0’ 5’ 30’ NC WCL 30’ 0’ 5’ NC Akt1 PKC ζ PKC ζ Akt1

Akt2 Plays a Critical Role in Metastasis Contro siAkt2/MDA �� 14 l EGF (ng/ml) 0 30’’ 1’ 0 30’‘ 1’ 2’ 5’ 2’ 5’ 12 0 p-LIMK 1 10 Chemotaxis Index 10 p-cofilin 100 8 cofilin 6 4 2 0 Control C44 C97 C102 Control � C44 � C98 � C97 � C102 � Akt2 Akt1

PDK1 is Required for Metastasis

An Optimal Level of PTEN is required for Chemotaxis � 1 2 V5-His-PTEN Clone100 Clone 74 Clone 6 PTEN Control M DA �� EGF (ng/ml) 15 0 �� Chemtoaxis Index PTEN 1 10 GAPDH 100 10 1000 EGF (ng/ 0 12 ml) 5 Chemtoaxis Index 1 10 100 1000 8 0 MDA-MB-231 V5-His-PTEN/ ���� MDA-MB-231 4 PDK1 Control siPTEN/MDA 0 MDA Control Clone Clone Clone 6 74 100

������������������ TXNL2 ! ROS ! GSH ! NFkB ! metastasis JCI 2010

What’s Next? Co-immunoprecipitation MDA-MB-231 cell HEK293 cell

P32 Golgi(Giantin) scr � scr sip32 � sip32

B23 SiB ��� SCR SCR 20min 6h siB23

cPLA2a �

Treatments with Gd@C82(OH)22 Inhibit hepatoma growth in a mouse model 0.28 mg/kg 1.20mg/kg � Gd@C 82 (OH) 22 inhibits tumor Nano Lett Vol 5, pg 2050 growth in a breast tumor model. �

Gd@C 82 (OH) 22 induced iDC maturation and TH-1 response. ACS Nano 2010

Treatments with Gd@C82(OH)22 cancer cell chemotaxis and metastasis Metastasis Treatments � Rate � Saline 66.7% q.d. × 20 day 10.00 0 uM Chemotaxis Index C 60 (C(COOH) 2 ) 2 8.00 1 uM (0.4 mg/kg, n = 34.2% 10 uM 6.00 10) 4.00 C 60 (OH) 20 (0.4 38.0% mg/kg, n = 10) 2.00 Gd@C 82 (OH) 22 � 0.00 0 1 10 100 1000 0.35mg/kg 4.3 % q.d. × 20 day [EGF] (ng/ml) �

Summary 1. Treatment with Gd@C82(OH)22 inhibits tumor growth without detectable toxicity. 2. Gd@C 82 (OH) 22 doesn’t show cytotoxicity. 3. Gd@C 82 (OH) 22 inhibits blood supply to tumor tissues. 4. Gd@C 82 (OH) 22 induced tumor immunity. 5. Gd@C 82 (OH) 22 inhibits cancer cell chemotaxis Blood supply � Tumor immunity � Metastasis �