Transcript Diapositiva 1

12th Annual CTOS Meeting 2006
544.
545.
549.
563.
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614.
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720.
733.
734.
747.
748.
764.
Serum TRACP5B level in OS
Stromal cell of GCT
Gene expression in sarcoma
p63 in GCT
Senescence in Ewing sarcoma
Gene expression of metastatic OS
PARP-1 in Bone tumors
PBF in Ewing sarcoma
IHH signal in OS
12q amplification in OS
Multi-directional differentiation of OS
Transformation of MSC
CLDN7 in SS
EXT mutation and phenotype
p16 in MSC
EGFR in SS and MPNST
Two-hits of EXT mutation
Zyxin and CARP-1 in apoptosis
Tibial psedoarthrosis of NF1
FRZB in sarcomas
Immunologic assessment of allograft
MDM2 in sarcoma
S. Avnet
M. Salerno
D.E. Joyner
R. Kandel
K. Tanaka
J.W. Lisle
A. Franco
H. Yabe
W. Lo
S. Mejia-Guerro
S. Ohtsuka
Y. Shima
Y. Kohno
E. Pedrini
K. Shibata
D.G. Thomas
S. Capponcelli
M.C. Beckerle
D. Viskochil
Y. Guo
K. Nelson
S. Bauer
Bologna
Bologna
Salt Lake City
Toronto
Fukuoka
Syracuse
Montreal
Tokyo
Toronto
Toronto
Kyoto
Kyoto
Kyoto
Bologna
Kyoto
Michigan
Bologna
Salt Lake City
Birmingham
Los Angels
Seattle
Boston
12th Annual CTOS Meeting 2006
544.
545.
549.
563.
585.
614.
642.
671.
672.
679.
686.
689.
697.
698.
700.
704.
720.
733.
734.
747.
748.
764.
Serum TRACP5B level in OS
Stromal cell of GCT
Gene expression in sarcoma
p63 in GCT
Senescence in Ewing sarcoma
Gene expression of metastatic OS
PARP-1 in Bone tumors
PBF in Ewing sarcoma
IHH signal in OS
12q amplification in OS
Multi-directional differentiation of OS
Transformation of MSC
CLDN7 in SS
EXT mutation and phenotype
p16 in MSC
EGFR in SS and MPNST
Two-hits of EXT mutation
Zyxin and CARP-1 in apoptosis
Tibial psedoarthrosis of NF1
FRZB in sarcomas
Immunologic assessment of allograft
MDM2 in sarcoma
S. Avnet
M. Salerno
D.E. Joyner
R. Kandel
K. Tanaka
J.W. Lisle
A. Franco
H. Yabe
W. Lo
S. Mejia-Guerro
S. Ohtsuka
Y. Shima
Y. Kohno
E. Pedrini
K. Shibata
D.G. Thomas
S. Capponcelli
M.C. Beckerle
D. Viskochil
Y. Guo
K. Nelson
S. Bauer
Bologna
Bologna
Salt Lake City
Toronto
Fukuoka
Syracuse
Montreal
Tokyo
Toronto
Toronto
Kyoto
Kyoto
Kyoto
Bologna
Kyoto
Michigan
Bologna
Salt Lake City
Birmingham
Los Angels
Seattle
Boston
12th Annual CTOS Meeting 2006
t(11;22)(q24;q12) results in EWS-Fli1 fusion gene in Ewing’s sarcoma.
Inhibition of EWS-Fli1 expression by antisense oligo causes G1 arrest in the cell cycle
in Ewing’s sarcoma cells.
Sense
(%)
80
(%)
Antisense
80
S
60
40
G1
G2+M
0
0
20
40
S
40
20
20
G1 arrest
G1
60
G2+M
Tanaka K, J Clin Invest,1997
0
0
20
60
80
Time after treatment (hr)
40
60
80
EWS-Fli1 promotes G1/S transition via unregulated expression of
Cyclins and CDK inhibitors in Ewing’s sarcoma cells.
Matsunobu T, Clin Cancer
Res,2004
Nakatani F, J Biol Chem,2003
Matsumoto Y, Br J Cancer,2001
Li X, Int J Cancer,2005
PURPOSE of the study is
to knockdown EWS-Fli1 expression by small-interfering RNA (siRNA)
for further elucidation of the function of EWS-Fli1
in oncogenesis of Ewing’s sarcoma.
12th Annual CTOS Meeting 2006
EWS-Fli1 causes p27 protein degradation probably via Skp2,
and evades senescence in Ewing’s sarcoma cells.
G1 arrest
Senescence
Skp2
EWS-Fli1
polyubiquitination of p27 protein
P
p27
T187
p27
degradation
P
T187
p27
Cyclin E
Cyclin E
CDK2
CDK2
26S proteasome
Cyclin E
CDK2
P
P
RB E2F
RB
P
E2F
G1/S
transition
12th Annual CTOS Meeting 2006
GENE EXPRESSION PROFILING IN METASTATIC
OSTEOSARCOMA BY cDNA MICROARRAY
Jennifer Lisle, Maria Iannolo, Matthew Allen, Jason Horton, Timothy Damron
SUNY Upstate Medical University
Syracuse, New York
• INTRODUCTION:
•
Purpose: To investigate gene
expression differences in a low
versus high metastatic human
osteosarcoma cell line
• METHODS:
•
•
•
SaOS LM2 (low) vs LM7 (high)
cDNA microarray differential
expression analysis
RT-PCR confirmation
12th Annual CTOS Meeting 2006
GENE EXPRESSION PROFILING IN METASTATIC
OSTEOSARCOMA BY cDNA MICROARRAY
• FINDINGS:
• CONCLUSIONS:
•
Some genes previously reported in OGS
literature assoc’ed w/ higher metastatic
cell line
– integrin β2, MME, l/b/k
ALP, S100A4
•
Others novel
– COL11A1, TM4SF10,ILR1
– PACE-1 (ezrin modulator)
•
Due to variability w/ in vitro techniques,
needs validation w/ in vivo model
(work underway)
12th Annual CTOS Meeting 2006
Expression of claudin7 is tightly associated with epithelial structures in
synovial sarcomas, and regulated by an Ets family transcription factor, ELF3.
Yoshiki Kohno1,2, Tatsuya Ishibe1,2, Satoshi Nagayama3, Koichi Nishijo1,2,
Yasuko Shima1,2, Kotaro Roberts Shibata 1,2,Tomoki Aoyama1, Tomitaka Nakayama2,
Takashi Nakamura2, Junya Toguchida1
1. Inst. Frontier. Med. Sci, 2. Dept. Orthop. Surg. and
3.Surg. Surgical Basic Sci, Grad. School Med., Kyoto University
Introduction and Aim of the Study
Biphasic synovial sarcoma
Synovial sarcoma(SS) is a malignant mesenchymal
tumor with epithelial components, but the mechanism
of formation of them is still unclear. We analyzed
claudins which is one of the molecules forming the
tight junctions indispensable for epithelial structures
to uncover the mechanism of epithelial components of SS.
Materials and Methods
1.Materials
1) Tumor samples: 17 SS (8 monophasic and 9 biphasic SS)
2) Cell lines: 6 SS cell lines and 8 other cell lines
2.Methods
1) Expression analysis: RT (Reverse Transcription)-PCR
Which claudins among 23 members?
Quantitative RT-PCR (QRT-PCR)
How are they regulated?
Immunohistochemistry
2) Promoter analysis: Luciferase assay
3) DNA-protein binding analysis: Electrophoretic Mobility Shift Assay (EMSA)
Chromatin Immunoprecipitation (ChIP) assay
4) Ectopic expression with the transient transfection
5) RNA interference method
12th Annual CTOS Meeting 2006
Results
Expression of CLDN4, -7, and -10 in biphasic SS tumor (Immunohistochemistry)
CLDN4
CLDN7
CLDN10
Association of ELF3 with CLDN7 – Immunohistochemistry in biphasic SS
ELF3
Conclusions
•
•
•
CLDN4, -7 and -10 were identified as epithelial structurerelated CLDNs in biphasic SS, among which CLDN7 was
most specific.
Expression of ELF3 was closely associated with the
expression of CLDN7.
ELF3 positively regulated the transcription of CLDN7
through the binding to the ets site at-150.
CLDN7
Ets site at -150 is critical for
the up-regulation of the
CLDN7 promoter activity by
ELF3 - Luciferase assay
12th Annual CTOS Meeting 2006
OVEREXPRESSION OF FRZB, A SECRETED WNT ANTAGONIST, DECREASES
INVASION, MOTILITY AND TUMORIGENESIS IN SOFT TISSUE SARCOMAS
Yi Guo1; Xiaolin Zi1; Zach Koontz1; Alison Kim1; Jun Xie1; William Tap2; Fritz C. Eilber2; Bang H. Hoang1.
1University of California, Irvine, CA; 2UCLA Geffen School of Medicine, CA, United States.
Objectives: To determine whether FrzB, a secreted Wnt antagonist, has an anti-tumor effect on
soft tissue sarcomas, using HT-1080 cell line (fibrosarcoma) and SW872 cell line (liposarcoma) as
models.
Methods: mRNA level of FzrB in fibroblast, stromal and soft tissue sarcoma (STS) cell lines was
determined by real-time PCR. FrzB expression plasmid was transfected into HT-1080 and SW872
cells. Stable clones, HT1080/FrzB and SW872/FrzB, were selected with G418. The activity of
canonical Wnt signaling in transfectants was tested by TOPFLASH luciferase reporter assay. Nude
mouse models were used to examine in vivo tumorigenesis and lung metastasis activity. Cell
migration and invasion were evaluated by scratch wound assay and Matrigel assay respectively. To
elucidate the potential mechanism, epithelial-to mesenchymal transition (EMT) markers were
examined by western blot and real time PCR. Given the important role of c-Met/HGF in tumor cell
growth and metastasis, the expression of Met and its downstream targets AKT and MAPK were
also examined.
12th Annual CTOS Meeting 2006
Results: In contrast with the fibroblast and stromal cells, FrzB mRNA level in four soft tissue
sarcoma cell lines (HT1080, SW872, SK-LMS-1, Syn-1) was significantly down-regulated. Using
HT1080 cells and SW872 cells as models, the over-expression of FrzB in these cells inhibited the
canonical Wnt signaling, which was verified by TOPFLASH luciferase reporter assay. In a nude
mouse model, FrzB significantly suppressed the subcutaneous growth of HT1080 cells. Matrigel
assay and scratch wound assay showed that blocking Wnt signaling by Frzb significantly reduced
the invasive activity and motility of both cell lines. In a lung metastasis model, HT1080/FrzB formed
fewer lung nodules than control cells. This decrease in tumorigenesis, invasive activity and motility
may result from the inhibition of c-Met/HGF pathway by FrzB. In both cell lines, FrzB downregulate Met expression, resulting in the decreased phosphorylation of AKT and MAPK, both are
markers of tumor progression. In SW872/FrzB cells, decrease in invasive activity may in part be
related to the reversal of EMT. This is verified by up-regulated epithelial markers such as Ecadherin, keratin 8, keratin 18 and by down-regulated mesenchymal markers such as N-cadherin,
vimentin and fibronectin.
Conclusions: Our data demonstrates the down-regulation of FrzB in a subset of soft tissue
sarcomas. Blocking Wnt signaling by FrzB in STS resulted in decreased tumorigenesis, invasive
activity and motility. This is the first study to show that the anti-tumor activity of FrzB may be
through inhibition of the c-Met/HGF pathway. Moreover, in some subsets of STS, the antitumor
activity is associated with the reversal epithelial mesenchymal transition (EMT). Blocking Wnt
pathway by FrzB may represent novel therapeutic strategies for STS. Further experiments are
under way to test these observations and the potential mechanism in additional subtypes of
sarcomas.