Background: Sarcomas constitute a group of diverse diseases with many subtypes and varying histologies and with adverse prognosis using current chemotherapies. Osteosarcoma is a frequent sarcoma with low rate of response to current therapy due to inherent chemoresistance. Hence, new approaches for therapy involving targeting of specific survival pathways are needed.

Methodology: We used 17-AAG to block HSP90 and rapamycin (Ra) to block mTOR, in 2 osteosarcoma cell lines, HOS and KHOS. Cells were treated for 24 and 48 hours with 200 nM of 17-AAG or 5 micromolar of Ra. Gene Profiling (GP) was performed using the h-U133 2 Plus Array (Affymetrix). Results were pre-filtered using the GCOS pair-wise comparisons. ANOVA was filtered for >2-fold change between two conditions in a set. GPs were compared between control HOS or KHOS vs. 24 H or 48 H treatment with Ra or 17AAG. ANOVA of drug-treated sets were filtered using a p-value < 0.01. Gene identity, ontology, z-scores and KEGG metabolic pathways were derived. Proteomics was performed using triplicate 2D gels and LC-MS/MS for each combination. Gels were scanned, calibrated for linearity and images analyzed by Nonlinear Dynamics 2006 software, PG240, and imagees were warped to creat matched overlays to a single reference (TT900 module). Spots were normalized by dividing each spots volume by the total density of all spots to give the "spot percentage". Spot percentages from 3 gels were averaged, and ratios determined for the various comparisons.

Results and conclusions: 17-AAG is a good inducer of apoptosis in both sarcoma cell lines (IC50 of 50nM); Ra is a weak inducer of apoptosis (IC20 at 5 micromolar; see additional poster by Gazitt et al.). Gene Microarray of 17AAG treated HOS cells affected >1,000 and 1,600 genes at 24 H and 48 H respectively, with ~700 genes affected at both time points. In contrast, Ra affected 395 and 352 genes at 24 and 48H, respectively, with only 20 genes affected at both time points. Interestingly, >90% of the >2-fold increases/decreases were common in HOS and KHOS. Of the genes upregulated by 17AAG, >450 were in the Ontology of cell and protein metabolism (Z-score 3- 4.5); >50 genes were in response to proteins and chemicals (Z=2.5-14.0); >50 genes in chromatin modifications and >40 in regulation of cell cycle (Z=2-5, for both). Specifically, significant changes were observed in histones and histone deacetylases; cytoskeleton proteins; extracellular matrix proteins and metalloproteinases; heat shock proteins (HSPs); regulators of the MAP kinase, mTOR, IGF and VEGF pathways and regulators of cell cycle and apoptosis. A great number of effects detected by genomics (e.g. apoptosis, cell cycle, HSPs, contractile and ubiquitin proteins were confirmed by proteomics and by WB with several new targets discovered. Effect of Ra on gene expression were very modest and will be discussed in details in the poster.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA