Background: Neuroblastoma is one of the most frequent solid tumor in children, which accounts for 15% of childhood cancer death. More than half of patients experienced tumor relapse driven by cancer stem cells that can be isolated as spheres. DENN domain proteins serve as GDP/GTP-exchanging factor of Rab family small G protein (DENN/RabGEFs) and are implicated in the progression of several cancers possibly through the regulation of intracellular membrane traffic. However, their involvement in the progression of neuroblastoma has not been elucidated.
Methods: Spheres of neuroblastoma BE(2)-C cells were grown in sphere medium with a non-adherent dish. Total RNA was extracted from parental cells and spheres of BE(2)-C cells and mRNA expression of 18 DENN/RabGEFs isoforms was analyzed by real time RT-PCR. Overexpression and knockdown of DENND2A in BE(2)-C cells were achieved by stably transfecting DENND2A cDNA and shRNA, respectively, and subjected to the sphere formation, colony formation and xenograft formation assays. The correlation of DENND2A expression with overall survival probabilities of neuroblastoma patients was analyzed by the bioinformatics program R2 (http://r2.amc.nl).
Results: Among 14 DENN/RabGEFs expressed in BE(2)-C cells, DENND2A showed the most profound difference between spheres and parental cells. Knockdown of DENND2A significantly promoted the sphere, colony and xenograft tumor formation, whereas they were significantly suppressed by overexpression of DENND2A. Furthermore, high DENND2A expression was significantly associated with high overall survival probabilities of neuroblastoma patients.
Conclusion: These results suggest that DENND2A is involved in the progression of neuroblastoma.