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.

Ral signaling pathway in health and cancer

“…While the level of RalA‐GTP was elevated in MPNST cells as compared with nonmalignant Schwann cells, the expression of RalA and its activation were found to be higher in CD133+ MPNST‐enriched cell population in comparison to CD133‐depleted population . Finally, medulloblastoma CD133+ cells were also found to contain higher levels of RalA activation .…”

“…Rals regulate tumorigenesis and cancer progression in three ways: (1) through activation of Ral effector proteins such as RalBP1 and kinase Aurora A, (2) via activation of several signaling pathways such as phosphaolipase D1, Src, JNK, NF‐kB, and cyclin D, and (3) by phosphorylation of Ral proteins . Ral activation was shown to be involved in a number of different tumor types such as lung , colorectal , melanoma , pancreatic , squamous cell carcinoma , hepatocellular carcinoma , prostate , ovarian , bladder , chronic myelogenous leukemia , peripheral nerve sheath tumors , and medulloblastoma . Studies have even been completed showing Ral‐A autoantibodies as a potentially useful serum biomarker for prostate adenocarcinoma .…”

RalB directly triggers invasion downstream Ras by mobilizing the Wave complex

“…In almost all cancer types examined (pancreas, colon, lung, bladder, prostate, melanoma), increased overexpression and/or activation of both RalA and RalB have been observed in patient tumor samples compared with normal tissues, regardless of their Ras mutation status ( Yan and Theodorescu, 2018 ). Moreover, Ral-GTP level were found elevated in various tumor-derived cell lines harboring different Ras status, including pancreas ( Lim et al, 2005 ), colon ( Martin et al, 2011 ), bladder ( Saito et al, 2013 ), liver ( Ezzeldin et al, 2014 ), lung ( Male et al, 2012 ) and brain ( Ginn et al, 2016 ). The only exception so far is squamous cell carcinoma (SCC), where RalA was found to suppress rather than promote tumor progression ( Sowalsky et al, 2010 ).…”

The Interplay between Oncogenic Signaling Networks and Mitochondrial Dynamics

“…Activated Ras binds to RalGEFs, which promote the activation of the Ral GTPases RalA and RalB. Increased Ral activity has been reported in human pancreatic, bladder, and colon cancer cell lines and tissues [88,89,90,91] and RalA is required for the tumorigenic growth of many Ras-driven cancer cell lines [88,92]. Like MAPK and PI3K, RalA can also drive changes in mitochondrial morphology as RalA promotes activation and mitochondrial recruitment of Drp1 during mitosis [93].…”