session 9 - kartik krishnan

20
High Expression of Ezrin, a Determinant of Metastatic Behavior, in Ewing’s Sarcoma Kartik Krishnan, Gaurav Khanna, Stephen Hewitt, Chand Khanna, and Lee J. Helman Pediatric Oncology Branch National Cancer Institute National Institutes of Health Bethesda, Maryland USA

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High Expression of Ezrin, a Determinant of Metastatic Behavior,

in Ewing’s Sarcoma

Kartik Krishnan, Gaurav Khanna, Stephen Hewitt, Chand Khanna, and Lee J. Helman

Pediatric Oncology BranchNational Cancer InstituteNational Institutes of HealthBethesda, Maryland USA

0 25 50 75 100 1250.0

0.2

0.4

0.6

0.8

1.0

Days

Low Ezrin

Intermediate Ezrin

High Ezrin

Mice

Dogs Humans

Ezrin Expression Correlates with Survival in Osteosarcoma

Khanna C, et al., Nature Medicine, 10 (2); 2004

Ezrin Expression in Ewings Sarcoma

IB: Mouse MAb α−Ezrin (Sigma)

WV

VW LD

KA

D

TC

71

RD

ES

5838

PrimaryCultures

CellLines

202

96

66

45

VW 18 y.o. female presented in 1/01 with widely disseminated disease involving multiple bony sites including femurs, vertebrae, and skull. Diagnosed with Ewing’s sarcoma

with Type IV translocation. Progressed on chemo and radiation therapy. Died 8/01.

VWCell linePrimary xenograft developed tumors rapidly. Stable tissue culture growth established at approximately one year, confirmed presence of type IV EWS-Fli1 translocation.

In Mice, orthotopic implantation resulted in tumors with a latency between 21 and 56 days. Experimental metastases developed with intravenous injection between 45 and 80 days.

EZRIN

Membrane-cytoskeleton linker protein

Initially identified as a determinant of cell shape

Plays a critical role in cell motility and attachment

Activatation initiated by threonine phosphorylation at position 567

Subsequent tyrosine phosphorylation allows for interaction with signal transduction pathways

Band 4.1 homology α-helixN C

Y143 T567Y353

F-actinBinding

EZRIN T567A

UnselectedBatch

WB: α-Ezrin

WT Ezrin

EzrinT567A-GFP

SelectedClones

VW

VW

-A

VW

-GFP

WT

T567A

Dominant Negative Clones

CMV GFP

Bjornsti and Houghton Nat Rev Cancer

Cyclin D1

MYC

HIF 1α

Ezrin

AKT

IB: α−AKT

IB: α−P-Ser473 AKT

Serum: - + - +

VW

-GF

P

VW

-GFP

Ezr

in T

567A

4EBP1

SerumStimulation

(minutes)0 30 120 0 30 120

VW

-GFP

VW

-GFP

Ezr

in T

567A

IB: α-P-Thr37-Thr46-4EBP1

IB: α-4EBP1

p42/44 MAPK

Serum: - + - +

VW

-GF

P

VW

-GFP

Ezr

in T

567A

IB: α−P-Thr202Tyr204 p42/44

IB: α−p42/44

Mice

1 X 106 cells injected IV

Assessed for development of experimental metastases

At Day 70:

VW-GFP 3 / 6VW-GFP-EzrinT567A 0 / 6

Conclusions

1. Ezrin is highly and ubiquitously expressed in Ewing’s sarcoma.

2. Expression of a Ezrin containing a non-phosphorylatable mutation at threonine 567 functions as dominant negative.

3. These dominant negative effects are observed on AKT, as well as 4EBP1.

4. Ewing’s sarcoma cells expressing a dominant negative Ezrin form experimental metastases at a lower rate than wild type.

Future Directions

1. Expression of Ezrin anti-sense to completely remove Ezrin expression and evaluate growth, survival, and signaling.

2. Analysis of effect of dominant negative and anti-sense Ezrin in other Ewing’s sarcoma cell lines, each with a different base line level of Ezrin expression.

3. Orthotopic implantation of dominant negative expressing cell lines, followed by amputation in mice to evaluate metastatic potential of these cells.

4. Further molecular studies:a. What other signal transduction pathways involve Ezrin?b. What are the Ezrin kinases? Are they suitable targets for

small molecule inhibition?c. Proteomics

Acknowledgments

Lee HelmanChoh YeungMelissa BurgosShaan Gandhi

Pediatric Oncology BranchNational Cancer Institute, NIH

Tumor and Metastasis Biology Section Pediatric Oncology Branch National Cancer Institute, NIHChand KhannaGaurav KhannaArnulfo Mendoza

Tissue Array Research Project National Cancer Institute, NIHStephen Hewitt

Murine Microarray Analysis:

Khanna C, et al., Cancer Research, 61 ;2001

Highly Metastatic K7M2 Primary Tumor

Less Metastatic K12 Primary Tumor

VS

EZRIN

K7M2 Wild type

K12 Wild type

0 25 50 75 100 125

0.0

0.2

0.4

0.6

0.8

1.0K7M2-wt

K12

K7M2-13

K7M2-1.46

K7M2-1.52

K7M2-2.13

K7M2-2.12

K7M2-2.15

Days

Ezrin

K7M

2 w

t

K12

wt

K7M

2/A

S E

zrin

13

K7M

2/A

S E

zrin

1.4

6

K7M

2/A

S E

zrin

1.5

2

K7M

2/A

S E

zrin

2.1

2

K7M

2/A

S E

zrin

2.1

3

K7M

2/A

S E

zrin

2.1

5

Tubulin

Relative Expression 21.

31.0 12.4 1.1 1.1 12.2 15.9 2.8

mTOR Signaling Pathway (PIK-related kinase, related to ATM, TRRAP etc.)

Bjornsti and Houghton Nat Rev Cancer

Osteosarcoma

Ezrin

P

P

Threonine 567Phosphorylation

F-Actin

CD44, PDGFRA

Tyrosine

Phosphorylation

RhoGDI

p85PI3K

AKTGrowth,Survival

P

P