gms | German Medical Science

Objectives: Chondrosarcoma is the second most common form of primary malignant bone tumors overall, but is the most common form in adults. Accumulating evidence from recent studies point to a critical role of Sox9, the master transcription factor for chondrogenesis, as a proto-oncogene in plenty of different tumor types. Especially in chondrosarcoma and osteosarcoma, Sox9 is presumed to influence the development and progression of the tumors, as well as the overall survival rate of patients, however the molecular mechanism behind these effects are still unclear.

Methods: We have introduced a transient Sox9 knockdown using specific Sox9 siRNA into a human chondrosarcoma cell line (HTB 94). Functional assays to investigate apoptosis (Caspase 3/7), adhesion (crystal violet staining), migration (boydn chamber), invasion (boydn chamber containing matrigel), the ability to form colonies (colony forming assay) and the viability (WST-1 assay) in the presence of cytostatic drugs (doxorubicin, cisplatin) were performed with Sox9 knockdown cells. Gene expression analysis of the cell survival genes BCL-2 and Survivin, and of insulin-like growth factors 1 and 2 (IGF1, IGF2) were analyzed via quantitative real-time PCR.

Results and conclusion: A reproducible knockdown of Sox9 in HTB 94 cells on mRNA and protein level (80% - 90%) was achieved using siRNA. In Sox9 knockdown cells a significant increase in apoptotic activity and enhanced adhesion capacity was observed whereas migration ability was unaltered and invasion and colony forming capacity was decreased. Preliminary results indicate that the sensitivity to doxorubicin is increased in Sox9 knockdown cells compared to control cells, whereas the treatment with cisplatin did not reveal differences in cell vitality between Sox9 knockdown cells and control cells. Gene expression of the anti-apoptotic genes BCL-2 and Survivin was significantly decreased after Sox9 inhibition. Moreover, IGF2 but not IGF1 gene expression was significantly decreased.

Based on these studies, we conclude a critical effect of Sox9 especially on apoptosis and cell survival via Bcl-2- and Survivin-mediated mechanism, possibly also influencing the sensitivity to doxorubicin. As also other tumor-specific properties like adhesion, invasion and colony forming ability were affected by inhibiting Sox9, we assume a role of Sox9 as a pro-survival factor and a possible proto-oncogene for sarcoma cells, which could strongly influence the tumorigenic potential and might thus be conceivable as a prognostic factor. Probably Sox9 also influences the glucose metabolism in tumor cells as IGF2 gene expression was impaired, but further studies are needed to elucidate this mechanism in detail.