Background: Cancer stem cells (CSCs) are regarded as essential for tumor maintenance, recurrence and distant metastasis. However, CSC or tumor-initiating cells were not identified in neuroblastoma (NB). We previously reported that stem cell related gene CD133 was expressed in neuroblastoma (NB) spheres and controls tumor sphere formation (Takenobu et al., 2010).
Methods: Gene expression profiling of adherent (control) and sphere forming NB cell lines as well as primary NB cells were conducted by microarray and RNA seq using illumina next generation sequencer. CDX1 knockdown and over-expression were performed by lentiviral system.
Results: To study the regulatory mechanism of CD133 transcription in NB tumor sphere, we analyzed activity of the promoter region of CD133, and found a promoter region which mainly regulates CD133 transcription in sphere-forming NB cells. To identify the responsible transcription factors, we analyzed the promoter region by in Silico and wet-lab analysis and identified sphere specific gene CDX1. Intriguingly, CDX1 specifically bound to the sphere-specific CD133 promoter region and promoter regions for reprogramming factors, including OCT4 and NANOG; CDX1 also regulates sphere formation efficiency in NB cells as well as CD133. In NB sample analysis by expression microarray, CDX1 high expression related to the unfavorable prognosis. These results indicate that CDX1 plays a role in NB cell stemness and aggressiveness. We also performed that comprehensive analysis in CDX1-expressing NB cell and sphere forming NB cells to identify NB-CSC specific genes and signaling pathways. The pathway analysis and Gene Set Enrichment Analysis (GSEA) suggested that upregulated genes were enriched for ES-related transcription and developmental signaling pathways in both the CDX1-expressing and sphere-forming NB cells. Taken together, CDX1 regulates the stemness-related pathways and seems to be a target of cancer stem cell-targeted therapy development.