SOX9 is a survival factor in chondrosarcoma and contributes to polyploidy, drug resistance and tumor malignancy

Objectives Chondrosarcoma is the second most common form of primary malignant bone tumor overall, and the most common form in adults. Accumulating evidence from recent studies points to a critical role of Sox9, the master transcription factor for chondrogenesis, as a proto-oncogene in plenty of different tumor entities. 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 mechanisms underlying these effects are still unclear.

Methods Sox9 gene expression in chondrosarcoma biopsies (grade I, II, III) was analysed via qRT-PCR and Sox9 protein expression via immunohistochemistry in a custom made tissue-micro-array (grade III). 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). We analyzed the doubling time of Sox9 knockdown and knockout cells and determined the cell-cycle distribution via FACS. Functional assays to investigate apoptosis (Caspase 3/7), invasion (Boyden chamber with matrigel), migration (scratch assay) and the ability to form colonies (colony forming assay) were performed with Sox9 knockdown and knockout cells. Viability in the presence of cytostatic drugs (doxorubicin, cisplatin) and after treatment with an oncolytic virus (T-VEC) was measured with WST-1 assay. Expression analysis of the cell survival genes BCL-2, Survivin, p21 and p53 was analyzed via qRT-PCR and western blot, and MMP2 activity was determined via gelatin zymography.

Results and Conclusion Our study showed that SOX9 expression is reduced in high-grade (III) chondrosarcoma and, in addition, in the non-chondroid, malignant compartments of dedifferentiated chondrosarcoma. Crispr/Cas9 mediated knockout of SOX9 in HTB94 cells results in reduced proliferation, clonogenicity and migration. Contrary, invasion, apoptosis and genetic instability was favored, involving cell survival mediators like BCL-2, Survivin, p21 and p53. Transient siRNA mediated SOX9 knockdown partly confirmed these results, suggesting that a minimum SOX9 threshold is required for specific carcinogenic functions. To overcome the ineffectiveness of anticancer therapies in terms of chondrosarcoma, possible therapy approaches were analyzed in SOX9 knockout cells. The cytotoxic effect of doxorubicin was enhanced, but sensitivity for oncolytic virus treatment with T-VEC was reduced. Hence, we suggest that SOX9 functions both as a pro-survival factor in HTB94 cells and as a possible proto-oncogene, that way influencing tumor malignancy. Moreover, absence of SOX9 might be a requirement for tumor progression into higher grades or into a dedifferentiated state. Our observations present a novel insight into the role of SOX9 in chondrosarcoma and provide a strong rationale for targeting SOX9 as a novel treatment strategy for chondrosarcoma.

Stichwörter SOX9, chondrosarcoma, CRISPR/Cas9

Publication History

Article published online:
15 October 2020