SOX2 predicts local spreading and promotes a cancer stem cell-like phenotype in oral squamous cell carcinoma

Abstract

Abstract Background Emerging evidence points toward the idea that oral squamous cell carcinoma (OSCC) invasiveness can be attributed to the existence of a small subpopulation of cancer stem cells (CSCs) in the bulk of the tumor. However, knowledge about the existence of CSCs in the OSCC close margins and their possible role in determining OSCC phenotypes is still far from being satisfactory. Methods We analyzed the expression of 13 CSCs marker genes in 72 primary tissue specimens (24 core tumors, 24 pathologically negative close margins, and 24 healthy distant margins), obtained from 24 OSCC patients upon surgical resection, by using Real-Time PCR. TGCA and GEO databases were used to confirm gene expression analyses. CAL27 and SCC15 OSCC cell lines were used to perform in vitro siRNA-mediated knockdown of SOX2 both in 2D and 3D cultures. The effects of SOX2 knockdown on OSCC cell lines were assessed by wound healing assays and 3D tumor spheroids formation assays. Cytofluorimetric apoptosis assays were used to test the effects of SOX2 knockdown on cisplatin sensitivity. Live imaging was used to monitor OSCC cells migration and spheroidogenesis. Results We found that BMI1, CD44, SOX2, OCT4, UBE2C, CXCR4 were significantly up-regulated, while IGF1-R, KLF4, ALDH1A1, CD133, FAM3Cappeared down-regulated in the tumor core vs healthy distant margin. No differences were observed for NANOG and RRM2. SOX2, CD44, and CXCR4 were upregulated also in the close margin vs healthy distant margin. The high expression of SOX2 both in the tumor core and in the close margin significantly correlated with tumor size and lymph node compromise. In vitro experiments further showed that SOX2knockdown i) promotes the mesenchymal-to-epithelial transition and smooths the invasiveness, ii) attenuates the 3D tumor sphere-forming capacity and stemness-associated traits and iii) partially increases the sensitivity to cisplatin treatment of CAL27 and SCC15 cell lines. Conclusions Overall, our study shows that the OSCC close margins can retain CSC-specific markers even in the setting of negative histopathological diagnosis. In particular, SOX2 may represent a promising marker useful to predict a more aggressive OSCC phenotype and a suitable target to prevent local invasiveness.