e2f3 in soft tissue sarcoma development m. scurr; a feber; j shipley; a fletcher; n dennis; i...
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E2F3 in Soft Tissue Sarcoma
Development
M. Scurr; A Feber; J Shipley; A Fletcher; N Dennis; I Judson; C Cooper
Active E2F3
RB E2F3
P
P
Cyclin DCDK
RB
Cyclin E, CDK2 G1/S G1/S TransitionTransition
Growth Factors
P
DNA synthesis (TS,TK,DHFR)
Cyclin A
E2F3 Overexpression and Cancer
Human Bladder Cancer (Feber et al)
E2F3 an oncogene activated by gene amplification and overexpression.E2F3 overexpression correlates with increasing grade and stage Prostate Cancer (Foster et al)
E2F3 overexpression an independent variable overall survival (P=.0014)cause specific survival (P=.0004)
Evaluation of Potential Role for E2F3 in STS
RB1 mutation/loss in STS
~1/3 of leiomyosarcomas
Up to 70% of primary tumours altered RB expression
Reports of cyclin D1/Cdk4 overexpression
Implicating RB-E2F Axis
STS Tissue Microarrays
165 patients with primary tumour samples and clinical data
Tumour TypeTumour Type Patient NPatient Noo
Leiomyosarcoma 118
MFH 97
Synovial Sarcoma 52
Fibrosarcoma 23
TOTAL 290
Fibrosarcoma
MFH
Leiomyosarcoma
Monophasic Synovial Sarcoma
Epithelial cell staining Spindle cell staining
E2F3 Nuclear Staining:Total STS TMA
38%
53.6%
5.6%2% 0.7%
0% 1-20% 21-40% 41-60% 61-80% 81-100%
Leiomyosarcoma Fibrosarcoma
MFH Synovial Sarcoma
44.7%52.8%
2.4%
neg upto 20% 21-40% 41-60% 61-80% 81-100%
32%
64.1%
3.9%
neg upto 20% 21-40% 41-60% 61-80% 81-100%
50%45.8%
4.2%
neg upto 20% 21-40% 41-60% 61-80% 81-100%
29.1%
40%
16.4%10.9%
3.4%
neg upto 20% 21-40% 41-60% 61-80% 81-100%
Synovial Sarcoma Prostate Cancer
29.1%
40%
16.4%10.9%
3.4%
33.3% 31.3%
9.5%13.6%
6.8% 5.4%
Clinical Correlations: E2F3 Staining and Prognostic Variables.
• Size
• Stage
• Grade
Clinical Correlations: E2F3 Staining and Prognostic Variables.
• Size No significant correlation
• Stage No significant correlation
• Grade
Clinical Correlations: E2F3 Staining and Prognostic Variables.
• Size No significant correlation
• Stage No significant correlation
• Grade P<0.001
Clinical Outcomes: Disease Free Survival
UNIVARIATE ANALYSIS size
stagegrade
MULTIVARIATE ANALYSIS stage
+ve E2F3 nuclear stainingHR 1.41 (95% CI .89-2.44) P=0.148
Clinical Outcomes: Disease Free Survival
UNIVARIATE ANALYSIS size
stagegrade
MULTIVARIATE ANALYSIS stage
+ve E2F3 nuclear stainingHR 1.45 (95% CI 0.93-2.25) P=0.1
Clinical Outcomes: Cause Specific Survival
UNIVARIATE and MULTIVARIATE ANALYSESsizestagegradesite
Clinical Outcomes: Cause Specific Survival
UNIVARIATE and MULTIVARIATE ANALYSESsizestagegradesite
+ve E2F3 nuclear staining HR 1.57 (95% CI 0.92-2.7) P=0.10
Assessment of biological function of E2F3 overexpression in STS
E2F3 in STS Cell LinesH
T1
08
0
HTB
-88
HTB
-11
4
SK
W
SS-S
Y-I
I
BA
X
FUJI
56
37
(+ve
contr
ol)
GAPDH
E2F3
E2F3 in STS Cell LinesH
T1
08
0
HTB
-88
HTB
-11
4
SK
W
SS-S
Y-I
I
BA
X
FUJI
56
37
(+ve
contr
ol)
GAPDH
E2F3
E2F3 siRNA Transfection: 24 hours
E2F3
GAPDH
Si-
1
Si-
2
Si-
3
Si-
all
Scr
-1
Scr
-2
Scr
-3
Scr
-all
contr
ol
Biological effect of “knocking down” E2F3 production
Si-3 and Scr-3 transfection of HT 1080
Analysis of effect on G1/S transition
BrdU Incorporation:Immunofluorescence Microscopyfluorescent activated cell sorting (FACS)
BrdU Incorporation Immunofluorescence Microscopy
0%
5%
10%
15%
20%
25%
30%
35%
40%
45%
50%
0hrs 24hrs 48hrs 72hrs
Si-3
Scr-3
BrdU Incorporation: FACS
Si-3: 0 hrs Si-3: 24 hrs post
Scr-3: 0 hrs Scr-3: 24 hrs post
Conclusions
• 62% STS samples positive E2F3 nuclear staining• LMS/MFH and fibrosarcoma predominantly <20% intensity• Synovial sarcoma more intense staining similar pattern to prostate and
bladder cancer.
• +ve Nuclear staining correlates with higher grade• Non-significant trend to poorer disease free and cause specific survivals
• E2F3 knockdown studies demonstrate that inhibition of E2F3 expression inhibits cell cycle progression.
E2F3 OVEREXPRESSION HAS A ROLE IN THE MALIGNANT E2F3 OVEREXPRESSION HAS A ROLE IN THE MALIGNANT PHENOTYPE IN STSPHENOTYPE IN STS
AcknowledgementsInstitute of Cancer Research
Molecular CarcinogenesisColin CooperAndy FeberSandra EdwardsPenny FlohrToby RoeJeremy ClarkJanet ShipleyAnne FletcherNening Dennis
Cancer TherapeuticsJenny TitleyMike Walton
Royal Marsden Hospital
Sarcoma UnitIan JudsonOmar Al-Muderis
Histopathology DepartmentCyril Fisher
StatisticsRoger Ahern
University of LiverpoolDepartment of Pathology and Medical
GeneticsChris FosterAndy Dodson
Supported by: Royal Marsden NHS Trust Charitable Fund Grant
Jeannette Pomeraniec Sarcoma Research Fund