12 th Annual CTOS Meeting Serum TRACP5B level in OSS. AvnetBologna 545.Stromal cell of GCTM. SalernoBologna 549.Gene expression in sarcomaD.E. JoynerSalt Lake City 563.p63 in GCTR. KandelToronto 585.Senescence in Ewing sarcomaK. TanakaFukuoka 614.Gene expression of metastatic OSJ.W. LisleSyracuse 642.PARP-1 in Bone tumorsA. FrancoMontreal 671.PBF in Ewing sarcomaH. YabeTokyo 672.IHH signal in OSW. LoToronto q amplification in OSS. Mejia-GuerroToronto 686.Multi-directional differentiation of OSS. OhtsukaKyoto 689.Transformation of MSCY. ShimaKyoto 697.CLDN7 in SSY. KohnoKyoto 698.EXT mutation and phenotypeE. PedriniBologna 700.p16 in MSCK. ShibataKyoto 704.EGFR in SS and MPNSTD.G. ThomasMichigan 720.Two-hits of EXT mutationS. CapponcelliBologna 733.Zyxin and CARP-1 in apoptosisM.C. BeckerleSalt Lake City 734.Tibial psedoarthrosis of NF1D. ViskochilBirmingham 747.FRZB in sarcomasY. GuoLos Angels 748.Immunologic assessment of allograftK. NelsonSeattle 764.MDM2 in sarcomaS. BauerBoston

12 th Annual CTOS Meeting Serum TRACP5B level in OSS. AvnetBologna 545.Stromal cell of GCTM. SalernoBologna 549.Gene expression in sarcomaD.E. JoynerSalt Lake City 563.p63 in GCTR. KandelToronto 585.Senescence in Ewing sarcomaK. TanakaFukuoka 614.Gene expression of metastatic OSJ.W. LisleSyracuse 642.PARP-1 in Bone tumorsA. FrancoMontreal 671.PBF in Ewing sarcomaH. YabeTokyo 672.IHH signal in OSW. LoToronto q amplification in OSS. Mejia-GuerroToronto 686.Multi-directional differentiation of OSS. OhtsukaKyoto 689.Transformation of MSCY. ShimaKyoto 697.CLDN7 in SSY. KohnoKyoto 698.EXT mutation and phenotypeE. PedriniBologna 700.p16 in MSCK. ShibataKyoto 704.EGFR in SS and MPNSTD.G. ThomasMichigan 720.Two-hits of EXT mutationS. CapponcelliBologna 733.Zyxin and CARP-1 in apoptosisM.C. BeckerleSalt Lake City 734.Tibial psedoarthrosis of NF1D. ViskochilBirmingham 747.FRZB in sarcomasY. GuoLos Angels 748.Immunologic assessment of allograftK. NelsonSeattle 764.MDM2 in sarcomaS. BauerBoston

12 th 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. Tanaka K, J Clin Invest,1997 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.

12 th Annual CTOS Meeting S proteasome polyubiquitination of p27 protein Ub degradation E2F RB P P P E2F G1/S transition EWS-Fli1 p27 EWS-Fli1 causes p27 protein degradation probably via Skp2, and evades senescence in Ewing’s sarcoma cells. Cyclin E CDK2 P p27 T187 P p27 T187 Cyclin E CDK2 Cyclin E CDK2 Skp2 G1 arrest Senescence

12 th 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

12 th Annual CTOS Meeting 2006 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) GENE EXPRESSION PROFILING IN METASTATIC OSTEOSARCOMA BY cDNA MICROARRAY

12 th 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 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. 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 Quantitative RT-PCR (QRT-PCR) 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 Biphasic synovial sarcoma Which claudins among 23 members? How are they regulated? Materials and Methods Introduction and Aim of the Study

12 th Annual CTOS Meeting 2006 CLDN4, -7 and -10 were identified as epithelial structure- related 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. Results Conclusions CLDN4CLDN7CLDN10 Association of ELF3 with CLDN7 – Immunohistochemistry in biphasic SS ELF3CLDN7 Expression of CLDN4, -7, and -10 in biphasic SS tumor (Immunohistochemistry) Ets site at -150 is critical for the up-regulation of the CLDN7 promoter activity by ELF3 - Luciferase assay

12 th 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.

12 th 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 down- regulate 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 E- cadherin, 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.