Background: Neuroblastoma is one of the most aggressive pediatric tumor that is characterized by its biological and clinical heterogeneity. Despite the current intensive therapy, more than half of high-risk patients have experienced a tumor relapse. Neuroblastoma recurrence is primarily driven by chemoresistant cancer stem cells that can be grown as spheres. Although Rab family small G proteins (Rabs) are implicated in the progression of growing number of cancers, the impact of their pathophysiological role in the progression of neuroblastoma remains to be determined.
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 64 Rabs expression was analyzed by real time RT-PCR. Rab6B, MTMR5 and Rab28 shRNAs and cDNAs were expressed in BE(2)-C cells and analyzed for intracellular localization, cell proliferation, sphere formation, colony formation, and xenograft tumor formation activities.
Results: When mRNA expression of all 63 Rabs in neuroblastoma BE(2)-C cells was examined, 48 Rabs were detected. Rab6B expression was most strongly induced in spheres compared to parental cells. During sphere formation, the primary intracellular localization of Rab6B was changed from the Golgi to the late endosome. By using Mass spectrometry, we identified a novel Rab6B-binding protein, myotubularin-related 5 (MTMR5) that was originally isolated as a SET-domain binding protein and known to activate Rab28 resided at the late endosome. Whereas MTMR5 and Rab28 decreased the sphere, colony and xenograft formation activities of BE(2)-C cells, Rab6B increased them by competing with Rab28 for the binding to MTMR5.
Conclusion: These results suggest that Rab6B mediates the progression of neuroblastoma through the interaction with MTMR5.