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 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 -helixN C
Y143 T567Y353
F-actinBinding
EZRIN T567A
UnselectedBatch
WB: -Ezrin
WT Ezrin
EzrinT567A-GFP
SelectedClones
VW
VW
-A
VW
-GF
P
WT
T567A
Dominant Negative Clones
CMV GFP
Bjornsti and Houghton Nat Rev Cancer
Cyclin D1
MYC
HIF 1
Ezrin
AKT
IB: AKT
IB: P-Ser473 AKT
Serum: - + - +
VW
-GF
P
VW
-GF
PE
zrin
T56
7A
4EBP1
SerumStimulation
(minutes)0 30 120 0 30 120
VW
-GF
P
VW
-GF
PE
zrin
T56
7A
IB: -P-Thr37-Thr46-4EBP1
IB: -4EBP1
p42/44 MAPK
Serum: - + - +
VW
-GF
P
VW
-GF
PE
zrin
T56
7A
IB: P-Thr202Tyr204 p42/44
IB: 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