Effects of GLI3 knockdown in the maintenance of stemness in oral squamous cell carcinoma


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Maria Fernanda Rodrigues1,Lucyene Miguita2,Nathália Andrade2,Fabio Nunes2
1Nove de Julho University-Post Graduate Program in Biophotonics Applied to Health Science,2Oral Pathology, School of Dentistry, University of São Paulo

Abstract

Background

Oral squamous cell carcinoma (OSCC) is a very aggressive disease associated with poor prognosis. Previous studies have demonstrated that cancer stem cells (CSCs) actively participate actively in OSCC development, progression and resistance to conventional therapy. In addition, CSCs frequently show deregulated expression of normal stem cell signaling pathways thereby acquiring their distinctive abilities as self-renewal and asymmetrical division.

Method

The SCC4 and SCC9 OSCC cell lines were sorted based on CD44 expression. The CD44high and CD44low cellular fractions were immediately collected to evaluate the differentially expressed genes related to stem cell signaling pathaways using PCR Array technique. Next, we have investigated the effects of GLI3 knockdown on CD44 and ESA expression by flow cytometry, colony and sphere formation capability and in stemness-related gene expression by RT-qPCR.

Results

Overexpression of genes related to Notch, TGFβ, FGF, Hedgehog, Wnt and pluripotency maintenance signaling pathways was observed in CD44high cells in relation to CD44low cells. GLI3 knockdown was associated with a significant decrease in CD44high/ESAhigh and CD44high/ESAlow cellular fractions, number of spheres and colonies as well as with the down-regulation of CD44, POU5F1 (OCT4) and BMI1 genes. This down-regulation was accompanied by an increase in the expression of IVL and S100A9 genes.

Conclusion

This study demonstrates for the first time that GLI3 contributes to OSCC stemness and malignant behavior, and that it may be a possible target, either isolated or in combination with other drugs, for therapies based on CSCs in OSCC.