gms | German Medical Science

Objectives: Sox9 is the master transcription factor for chondrogenesis but is also involved in the regulation of proliferation, apoptosis and cell signaling cascades. Accumulating evidence from recent studies point to a critical role of Sox9 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 and a stable Sox9 knockout using CRISPR/Cas9 technology 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) and the ability to form colonies (colony forming assay) were performed with Sox9-knockdown and knockout cells. The capacity of Sox9 knockdown cells to differentiate along the chondrogenic lineage was analyzed via quantification of marker genes (qPCR). In addition, doubling time, viability and tendency to form aggregates of different Sox9-knockout clones were verified using a cell counter.

Results and conclusion: A reproducible knockdown of Sox9 in HTB94 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. Using CRISPR/Cas9, a complete knockout of Sox9 in HTB94 cells was generated and resulted in an increase in doubling time (47h) compared to control cells (35h) and a decreased aggregate rate (13%), whereas viability was unaltered. Sox9 knockout-cells showed increased apoptotic activity, adhesion capacity, invasion and migration. The colony forming ability was significantly decreased when Sox9 is missing. Sox9 knockout clones have a strongly reduced COL2A1 mRNA expression. Differentiation studies with Sox9 knockdown cells resulted in decreased expression of COL1A1, COL2A1 and integrin alpha 11(ITG11) during the first two weeks of chondrogenic differentiation, while in a later phase, increased expression of COL10A1 points to an acceleration of the transition into a hypertrophic status.

Based on these studies, we conclude a critical effect of Sox9 on apoptosis, cell-cell and cell-surface adhesion, invasion and colony forming ability as well as on cell doubling time in a human chondrosarcoma cell line. These data point to a role of Sox9 as a critical 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 expression alters the balance between self-renewal and differentiation capacity, what might be the central point of action for Sox9 in tumor cells.