RalA is overactivated in medulloblastoma.

ABSTRACT

Medulloblastoma (MDB) represents a major form of malignant brain tumors in the pediatric population. A vast spectrum of research on MDB has advanced our understanding of the underlying mechanism, however, a significant need still exists to develop novel therapeutics on the basis of gaining new knowledge about the characteristics of cell signaling networks involved. The Ras signaling pathway, one of the most important proto-oncogenic pathways involved in human cancers, has been shown to be involved in the development of neurological malignancies. We have studied an important effector down-stream of Ras, namely RalA (Ras-Like), for the first time and revealed overactivation of RalA in MDB. Affinity precipitation analysis of active RalA (RalA-GTP) in eight MDB cell lines (DAOY, RES256, RES262, UW228-1, UW426, UW473, D283 and D425) revealed that the majority contained elevated levels of active RalA (RalA-GTP) as compared with fetal cerebellar tissue as a normal control. Additionally, total RalA levels were shown to be elevated in 20 MDB patient samples as compared to normal brain tissue. The overall expression of RalA, however, was comparable in cancerous and normal samples. Other important effectors of RalA pathway including RalA binding protein-1 (RalBP1) and protein phosphatase A (PP2A) down-stream of Ral and Aurora kinase A (AKA) as an upstream RalA activator were also investigated in MDB. Considering the lack of specific inhibitors for RalA, we used gene specific silencing in order to inhibit RalA expression. Using a lentivirus expressing anti-RalA shRNA we successfully inhibited RalA expression in MDB and observed a significant reduction in proliferation and invasiveness. Similar results were observed using inhibitors of AKA and geranyl-geranyl transferase (non-specific inhibitors of RalA signaling) in terms of loss of in vivo tumorigenicity in heterotopic nude mouse model. Finally, once tested in cells expressing CD133 (a marker for MDB cancer stem cells), higher levels of RalA activation was observed. These data not only bring RalA to light as an important contributor to the malignant phenotype of MDB but introduces this pathway as a novel target in the treatment of this malignancy.