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RalA is overactivated in medulloblastoma

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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.

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Acknowledgments

Faris Farassati dedicates this work to the honor of “Nicolas Marion Battaglia”. This work was supported by funding from Flight Attendant’s Medical Research Institute (FAMRI) and Saint Luke’s Hospital-Marion Bloch Foundation to Faris Farassati and Midwest Cancer Alliance (MCA) grant to Kevin Ginn and Faris Farassati and Children’s Mercy Cancer Center Auxiliary Grant to Kevin Ginn. We wish to express our gratitude for cooperation of Dr. Ossama Tawfik and Marcia Pressly at department of pathology and the staff of KUMC flow cytometry core, Richard Hastings and Alicia Zeiger. Parts of the preliminary work in this manuscript were presented at 2010 Society for Neuro-Oncology Scientific Meeting and Education Day (Montreal, Canada) and 2010 Children’s Oncology Group Fall Meeting (Dallas, TX).

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  1. Sharad Mathur & Faris Farassati

    Present address: Research Service (151), Kansas City Veteran Affairs Medical Center & Midwest Biomedical Research Foundation-Saint Luke’s Marion Bloch Brain Tumor Research Program, 4801 E Linwood Blvd, F5-123, Kansas City, MO, 64128, USA

Authors and Affiliations

  1. Molecular Medicine Laboratory, Department of Medicine, University of Kansas Medical School, Kansas City, KS, USA

    Kevin F. Ginn, Ben Fangman, Kaoru Terai, Amanda Wise, Daniel Ziazadeh, Kushal Shah, Robyn Gartrell, Brandon Ricke, Kyle Kimura, Emma Borrego-Diaz & Faris Farassati

  2. Division of Hematology and Oncology, Children’s Mercy Hospital and Clinics, Kansas City, MO, USA

    Kevin F. Ginn

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Kevin F. Ginn, Ben Fangman, Amanda Wise and Daniel Ziazadeh are equal participation authors. Kaoru Terai, Kushal Shah, Robyn Gartrell, Brandon Ricke, Kyle Kimura and Sharad Mathur are equal participation authors.

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Ginn, K.F., Fangman, B., Terai, K. et al. RalA is overactivated in medulloblastoma. J Neurooncol 130, 99–110 (2016). https://doi.org/10.1007/s11060-016-2236-4

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