Abstract 319: Cholesterol inhibition reduces Hh mediated chondrosarcoma

Constitutive activation of Hh signaling is a common occurrence in chondrosarcoma(CSA). Gene profiling analysis showed that Gli transcription regulates genes that govern cholesterol homeostasis, which alters cholesterol accumulation in chondrocytes; a higher level of Gli-mediated transcription results in accumulation of intracellular cholesterol. Here we determined if targeting cholesterol-processing genes downstream of Hh signalling could be used as a novel treatment approach. With institutional review board approval, human CSA samples were obtained fresh from surgery. For in vitro studies, CSA explants of 2mmx2mm x2 mm cubic in size establihed as organ cultures. For in vivo studies, one million CSA cells were subcutanously injected into NSG mice. Cells from five CSAs were treated both in vitro and in vivo with a hedgehog inhibitor, Cur61414, a cholesterol inhibitor, Lovastatin, or both. In vitro, CSA explants were treated for 48 hrs at concentration of 20 μM of each drug. In vivo, mice were treated with 4.5mg/kg/day of Cur61414, Lovastatin, or both by intraperitoneal injection for 4 wks. At the end of treatment, the explants or xenografts were harvested and processed for further analysis. RT-PCR was used to meausre the expression of Hh and cholesterol target genes. Tumor size was meausred from the xenografts. Blockade of Hh signaling significantly decreased Gli1 gene expression by 30%, increased 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) expression for more than 300% indicating decreased intracellular cholesterol. Treatment with the cholesterol inhibitor Lovastatin increased expression of HMGCR for more than 500%. The combination of Hh and cholesterol blockage resulted in increased expression of HMGCR for more than 3400%. Analysis of chondrosarcoma xenografts in vivo showed a significnat decrease in tumor size with Lovastatin (32% decline), 3 folds reducation of Brdu(+) cells, and 2.4 fold increase of Caspase-3 + cells, treatment with Cur 61414 reduced the tumor growth by 5% with no significant reduction of Brdu(+) cells but 2.6 fold increase of Caspase-3 (+) cells . The combination treatment of lovastatin and Cur61414 on xenografts resulted in a significnat decrease in tumor size (32% decline), 3 fold reduction of Brdu (+) cells, but no significant changes of Caspase-3 (+) cells . These data suggest that cholesterol functions downstream of Hh signaling pathway in CSA. The more effective reduction in tumor growth with cholesterol inhibition compared