Abstract
BackgroundMedulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. In addition, dysregulated function and signaling by Eph family of receptors have been shown to impart pro-tumorigenic characteristics in this brain malignancy. In the current study, we investigated whether EphB2 knockdown in combination with radiation can alter invasiveness and decrease medulloblastoma tumor growth or viability in vitro.MethodsThe expression of EphB2 receptor was analyzed by immunohistochemistry and Western blotting. Microarray analysis and mRNA analysis was performed on medulloblastoma patient datasets and compared to the normal cerebellum. The radiosensitization effect following EphB2 knockdown was determined by clonogenic assay in human medulloblastoma cells. Effects of EphB2-siRNA in absence or presence of radiation on cell cycle distribution, cell viability, and invasion were analyzed by flow cytometry, MTT assay, trypan blue exclusion assay, xcelligence system, and Western blotting.ResultsWe observed that EphB2 is expressed in both medulloblastoma cell lines and patient samples and its downregulation sensitized these cells to radiation as evident by decreased clonogenic survival fractions. EphB2 expression was also high across different medulloblastoma subgroups compared to normal cerebellum. The radiosensitization effect observed following EphB2 knockdown was in part mediated by enhanced G2/M cell cycle arrest. We also found that the combined approach of EphB2 knockdown and radiation exposure significantly reduced overall cell viability in medulloblastoma cells compared to control groups. Similar results were obtained in the xcelligence-based invasion assay. Western blot analysis also demonstrated changes in the protein expression of cell proliferation, cell survival, and invasion molecules in the combination group versus others.ConclusionsOverall, our findings indicate that specific targeting of EphB2 receptor in combination with radiation may serve as an effective therapeutic strategy in medulloblastoma. Future studies are warranted to test the efficacy of this approach in in vivo preclinical models.
Highlights
Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties
We analyzed transcriptomic data performed on a cohort of medulloblastoma patients obtained from Children’s Hospital Colorado and found that EphB2 is significantly overexpressed in these samples compared to the non-malignant brain samples (Fig. 1a)
Variability can be seen across the different datasets, similar trends were observed with all the four medulloblastoma subtypes showing elevated expression of EphB2 compared to the normal cerebellum (Fig. 1b, c)
Summary
Medulloblastoma is one of the most common types of pediatric brain tumor characterized by the subpopulation of cells that exhibit high invasive potential and radioresistant properties. Morrison et al [4], reported that certain Eph receptor tyrosine kinases and ligands were aberrantly regulated in the migrating medulloblastoma cells versus core tumor cells. These migrating cells were characterized by upregulation of EphB2 as shown by qPCR analysis in DAOY medulloblastoma cells [4]. Sikemma et al [5], demonstrated increased invasion in EphB2-expressing medulloblastoma cell lines following ligand stimulation This effect was completely blocked upon shRNA-mediated knockdown of EphB2 [5]. Another group showed that EphB2 overexpression enhanced neurosphere cell migration and invasion and its targeted inhibition resulted in decreased invasion/migration in the GBM model [6]
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