SOX2 Maintains the Stemness of Retinoblastoma Stem-like Cells Through Hippo/YAP Signaling Pathway

Abstract Background: Cancer stem cells are responsible for tumor initiation and progression in various types of cancer, while, although the existence of retinoblastoma stem cells had been reported, the mechanisms supporting retinoblastoma stemness are poorly understood. In this study, a modified method for isolating retinoblastoma stem-like cells for mechanistic study was first established and an important mechanism underlying SOX2-drived retinoblastoma stemness was subsequently revealed.Methods: The retinoblastoma stem-like cells were isolated by single cell cloning in combination of examination of sphere-forming capacities. The stemness of isolated retinoblastoma stem-like cells were characterized by sphere-forming capacities and the expression of cancer stem cell markers. The SOX2 gene was overexpressed or knocked down by lentivirus system. The transcriptional regulation was identified by qRT-PCR, luciferase reporter, nuclear run-on and DNA pull down assay. Spearman analysis was employed for correlation analysis of genes in tumor tissues of retinoblastoma patients. Results: The isolated retinoblastoma stem-like cells exhibited significantly enhanced sphere-forming capacity and constantly higher levels of CD44, ABCG2, SOX2 and PAX6, but not CD133. SOX2 positively regulated the stemness of retinoblastoma stem-like cells as identified by gene manipulation technology. SOX2 directly binds to the promoters of WWTR1 and YAP1, transcriptionally activates WWTR1 and YAP1, and thereby activating Hippo/YAP signaling. Knockdown of WWTR1 or YAP1 partially abolished the effect of SOX2 on the stemness of retinoblastoma stem-like cells. Conclusion: An effective method for isolation of retinoblastoma stem-like cells was established. The mechanistic study demonstrated that SOX2, as a key deriver, maintains retinoblastoma stemness by activating Hippo/YAP signaling. Inhibition of Hippo/YAP signaling would be an effective strategy for human retinoblastoma caused by aberrant upregulation of SOX2.