CTGF promotes stem-like properties of head and neck cancer cells

Purpose: We investigate the role of connective tissue growth factor (CTGF/CCN2), a cytokine providing crucial impact in different types of human cancers, in regulating the stem-like properties of head and neck cancer cells. Methods: Different assays including real-time PCR analysis of stemness genes, flow cytometric analysis of putative stem cell markers, spheroid formation assay, soft agar clonogenecity assay were applied to examine the difference between head and neck cancer cells overexpressing CTGF or treated by rCTGF and control. Results: We demonstrated that Sox2, Nanog and Oct4, three of the most important stemness genes, were up-regulated in SAS treated by rCTGF or overexpressing CTGF. To confirm this finding, we tested the expression profile of stemness genes in another head and neck cancer cell line FADU, and a consistent finding was shown: upregulation of stemness genes was noted in FADU treated by rCTGF or overexpressing CTGF. We subsequently analyzed the putative stem cell markers in SAS treated by rCTGF or overexpressing CTGF versus control. It revealed that SAS treated by rCTGF and SAS-CTGF clones enriched the side population, CD44-positive population and ALDH activity, indicating the stem-like properties, of oral cancer cells significantly. Functional assays for evaluating the self-renew property showed a significant increase in spheroid-forming ability in FADU treated with rCTGF. Overexpression of CTGF also enhanced the clonogenecity in SAS. Conclusions: Excessive endogenous (overexpression) or exogenous (recombinant) CTGF both promote stem-like properties of head and neck cancer cells, and the genes responsible for stemness properties are upregulated correspondingly.