Transcript Session 11 — Molecular Biology for the Patients: Define New Targets?

12th Annual CTOS Meeting 2006
SESSION 11
Molecular biology for the patients:
Define new targets? Predict Outcome?
Moderators: Ole Steen Nielsen & Irene Andrulis
• 22 Posters – all worth seeing!
• Selected 4 posters:
– # 526 Ueda et al, WT1 in STS
– # 557 Hannay et al, VEGF in STS
– # 578 Keschman et al, PI3K/AKT
– # 579 Shor et al, SRC kinase
12th Annual CTOS Meeting 2006
# 526: Ueda et al: Prognostic significance of WT1 expression in STS
INTRODUCTION:
WT1 gene, first identified from Wilms’ tumor
patients, encodes a zinc finger transcription factor, regulating the genes
related to cell differentiation, proliferation and apoptosis, such as PDGFα, IGF-II, c-myc and bcl-2.
WT1 was originally categorized as a tumor-suppressor gene, but recently
has been emphasized of its oncogenic properties.
MATERIALS & METHODS:
•Tissue samples; 52 primary soft-tissue sarcoma (STS) frozen samples
& 13 normal soft-tissue samples (control)
• To analyze WT1 mRNA expression level using quantitative real-time
RT-PCR
& univariate/multivariate surival analysis according to various
clinicopathological factors including WT1 mRNA expression level.
AIM of the STUDY: To evaluate the prognostic implication of
WT1 mRNA expression in patients with STS.
12th Annual CTOS Meeting 2006
RESULTS:
The levels of WT1 mRNA expression in various STSs were significantly
higher than those in normal soft-tissue samples. No significant correlation
was observed between WT1 expression level and various clinicopathological factors.
The disease-specific survival of patients with high WT1 levels was found to
be significantly worse than those with low WT1 levels (p=0.0182).
Multivariate analysis further indicated that WT1 expression level is a
significant prognostic factor (RR 2.6; p=0.0488), independent of other
prognostic factors including age, histological grade, and distant metastasis
at presentation.
CONCLUSIONS:
WT1 mRNA is frequently overexpressed in various types of STS, and its
expression level is a significant prognostic indicator for STS patients.
These results strongly suggest that WT1 can be a candidate for a potent
molecular marker to predict patient prognosis, and a molecular target for
tumor-specific therapy against STS.
12th Annual CTOS Meeting 2006
# 557 Hannay et al:
Targeting soft tissue sarcoma-associated endothelial cell
chemoresistance
via VEGFR inhibition: A strategy to prevent tumor growth and
metastasis
Introduction, materials & methods, and aim of study
• To better elucidate the role of VEGF165 in soft tissue
sarcoma (STS) growth, metastasis and
chemoresistance we…
– Stably transfected leiomyosarcoma (SKLMS-1) and
rhabdomyosarcoma (RD) human STS cell lines with VEGF165 to
generate a panel of SK-SE, and RD-SE daughter clones.
– Assessed tumor growth, invasion, metastasis, angiogenesis, and
chemoresistance phenotypes of parental and daughter lines in vivo
in SCID mice, and HUVEC stimulation in vitro.
– Assessed the effect of VEGFR2 inhibition with DC101 anti-VEGFR2
mAb on STS growth, angiogenesis, metastasis, and response to
doxorubicin in parental and daughter lines in vivo, and HUVEC
stimulation in vitro.
12th Annual CTOS Meeting 2006
Results, discussion & conclusions
• We found that:
– VEGF165 transfected xenografts formed highly vascular tumors with shorter
latency, accelerated growth, enhanced chemoresistance, and increased
incidence of pulmonary metastases.
– Blockade of VEGFR2 signaling using DC101 anti-VEGFR2 mAb enhanced
doxorubicin chemoresponse; inhibiting tumor growth and decreased
pulmonary metastases without overt toxicity.
– Combined therapy reduced microvessel counts while increasing vessel
maturation index.
– VEGF overexpression did not impact on the sarcoma cells per-se, however,
CM from VEGF tranfectants caused increased endothelial cell proliferation,
migration, and chemoresistance.
– DC101 induced EC sensitivity to doxorubicin and suppressed the activity of
MMPs secreted by EC.
•
We conclude that VEGF is a critical determinant of STS growth and metastasis
and that STS chemoresistance, in our model, is a process induced by the
interplay between STS cells and tumor associated endothelial cells. STS growth
and metastasis can be interrupted by combined low dose doxorubicin and antiVEGFR2, a strategy that attacks STS associated EC.
12th Annual CTOS Meeting 2006
# 578: Keschman et al:
LY294002 induces apoptosis in sarcoma independent of the PI3K-AKT pathway
Introduction and Aim of Study
•
The PI3K-Akt signaling pathway regulates cell survival, proliferation,
angiogenesis, translation, and apoptosis, and is known to be altered in
sarcomas
•
LY294002 acts on the ATP-binding site of the PI3K enzyme, which inhibits the
PI3K-Akt binding complex
•
LY294002 has been used successfully to induce apoptosis in cancer cells, and
this has previously been ascribed to its PI3K-Akt blocking activity
•
The aim of this study was to investigate the effects of LY294002 on cell cycle
proliferation, cell growth, and apoptosis in leiomyosarcoma, osteosarcoma,
Ewing’s sarcoma, and high-grade undifferentiated sarcoma, and to determine
the pathway by which this inhibitor induces apoptosis in these cells
12th Annual CTOS Meeting 2006
Results and Conclusions
•
•
•
LY294002 and wortmannin both inhibit Akt phosphorylation, however LY294002
induces apoptosis in sarcoma cells much more effectively, in both a time and
dose-dependent manner
After 24 hour treatment with LY294002, cells were arrested in G0-G1 phase,
according to flow cytometry analysis
At higher doses and after 48 hours, cells treated with LY294002 were killed by
apoptosis, as evidenced by PARP cleavage, TUNEL staining, and DNA
fragmentation ELISA assay
1.0
Figure 1. LY294002 induces
apoptosis more effectively than
wortmannin, as evidenced by DNA
fragmentation of cell nuclei in
SKLMS-1 leiomyosarcoma cells.
Absorbance
0.8
0.6
0.4
0.2
0.0
control
•
10uM LY
50uM LY
0.5uM Wort
1uM Wort
These results indicate that LY294002 induces apoptosis in leiomyosarcoma,
osteosarcoma, Ewing’s sarcoma, and high-grade undifferentiated sarcoma by
some mechanism independent of the PI3K-Akt pathway
12th Annual CTOS Meeting 2006
# 579 Shor et al:
Action of the SRC kinase inhibitor Dasatinib on human sarcoma cell lines
Introduction and Aim of Study
•
Sarcomas frequently possess abnormalities in growth factor receptor signaling
pathways, including PDGF-R, c-KIT, c-MET and IGF1-R.
•
A common point of signal convergence downstream of many of these pathways
is Src kinase, the first oncoprotein identified as a solid tumor virus, Rous
sarcoma virus, which induces sarcomas in chickens.
•
Src is regulated by growth factors, cytokines, cell adhesion and antigen receptor
activation, and is involved in controlling a myriad of fundamental cellular
processes including cell proliferation, migration, invasion and survival.
•
Several studies have demonstrated the activity of dasatinib, a Src kinase
inhibitor, in epithelial tumor cell lines and early phase clinical trials have
established the safety and efficacy of dasatinib for treatment of imatinib-resistant
chronic myelogenous leukemia.
•
However, the responses and mechanisms of action of dasatinib in
mesenchymally-derived tumor cell lines have not been described previously.
•
The aim of this study was to evaluate the activity of dasatinib in sarcoma cell
lines and determine the role for Src kinase in the survival of sarcoma cell lines.
12th Annual CTOS Meeting 2006
Results and Conclusions
•
•
•
Src kinase is activated in sarcoma tissue and cell lines.
Dasatinib inhibits Src activation and downstream signaling, cell motility and
invasion in sarcoma cell lines and induces apoptosis in bone sarcoma cell lines.
Furthermore, siRNA to c-Src induces apoptosis in a subset of bone sarcoma cell
lines.
Table 1. Summary of response to dasatinib in human sarcoma cell lines.
Cell Line
•
Histology
pSrc(Y419)
Expression
IC50, nM
pSrc (Y419)
Migration
Induction of
Parp Cleavage, nM
Sa-OS 2
Osteosarcoma
++
46
65
30
LM-2
Osteosarcoma
++
26
35
30
LM-7
Osteosarcoma
++
68
24
100
U2OS
Osteosarcoma
++++
57
4.0
30
MG-63
Osteosarcoma
+++
28
58
N/R
SK-ES
Ewing’s Sarcoma
+++
11
4.0
30
TC-71
Ewing’s Sarcoma
+
3.1
4.0
30
SKLMS-1
Leiomyosarcoma
+++
46
44
N/R
HT1080
Fibrosarcoma
-
N/R
N/R
N/R
RD
Rhabdomyosarcoma
++++
26
23
N/R
RD|18
Rhabdomyosarcoma
++
45
29
N/R
A673
Rhabdomyosarcoma
+
50
277
N/R
Taken together, our results suggest that dasatinib will provide clinical benefit to
soft tissue and other sarcomas by preventing metastasis, which may be further
augmented in bone sarcomas by induction of apoptosis.