YB-1 dependent oncolytic adenovirus efficiently inhibits tumor growth of glioma cancer stem like cells

Klaus Mantwill,Dagmar Beier,Vroni Girbinger,Ulrike Naumann,Hermann Lage,Janina Seznec,Per Sonne Holm,Jürgen Schlegel,Pawel Surowiak,Michel Mittelbronn

doi:10.1186/1479-5876-11-216

Abstract

BackgroundThe brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. In brain CSC, the PI3-K/AKT and the RAS/mitogen activated protein kinase (MAPK) pathways are found to be activated. In consequence, the human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. This study determines the expression of YB-1 in glioblastoma (GBM) specimen in vivo and in brain CSC lines. In addition, the capacity of Ad-Delo3-RGD, an YB-1 dependent oncolytic adenovirus, to eradicate CSC was evaluated both in vitro and in vivo.MethodsYB-1 expression was investigated by immunoblot and immuno-histochemistry. In vitro, viral replication as well as the capacity of Ad-Delo3-RGD to replicate in and, in consequence, to kill CSC was determined by real-time PCR and clonogenic dilution assays. In vivo, Ad-Delo3-RGD-mediated tumor growth inhibition was evaluated in an orthotopic mouse GBM model. Safety and specificity of Ad-Delo3-RGD were investigated in immortalized human astrocytes and by siRNA-mediated downregulation of YB-1.ResultsYB-1 is highly expressed in brain CSC lines and in GBM specimen. Efficient viral replication in and virus-mediated lysis of CSC was observed in vitro. Experiments addressing safety aspects of Ad-Delo3-RGD showed that (i) virus production in human astrocytes was significantly reduced compared to wild type adenovirus (Ad-WT) and (ii) knockdown of YB-1 significantly reduced virus replication. Mice harboring othotopic GBM developed from a temozolomide (TMZ)-resistant GBM derived CSC line which was intratumorally injected with Ad-Delo3-RGD survived significantly longer than mice receiving PBS-injections or TMZ treatment.ConclusionThe results of this study supported YB-1 based virotherapy as an attractive therapeutic strategy for GBM treatment which will be exploited further in multimodal treatment concepts.

Highlights

The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence
Brain CSC lines R11, R28, R40, and R49 were obtained from patients with primary GBM as previously described [31] and were maintained as tumorspheres in stem cell-permissive DMEM-F12 medium supplemented with 20 ng/ml of each human recombinant epidermal growth factor (EGF; BD Biosciences, Heidelberg, Germany), human recombinant basic fibroblast growth factor, human leukemia inhibitory factor (LIF; Millipore, Billerica, MA, USA), and 2% B27 (Life Technologies, Carlsbad, CA, USA) for preservation of the tumors’ original molecular characteristics and for minor differentiation
Brain CSC lines express activated AKT and mitogen activated protein kinase (MAPK)/ERK It is established that AKT, MAPK/ERK and ribosomal S6 kinase (RSK) can interact with and phosphorylate YB-1 [37,38,39]

Summary

Introduction

The brain cancer stem cell (CSC) model describes a small subset of glioma cells as being responsible for tumor initiation, conferring therapy resistance and tumor recurrence. The human transcription factor YB-1, knowing to be responsible for the emergence of drug resistance and driving adenoviral replication, is phosphorylated and activated. With this knowledge, YB-1 was established in the past as a biomarker for disease progression and prognosis. Considerable research efforts have been focused on dissecting the role of cancer stem cells (CSC) referred to as tumor initiating cells (TIC), in cancer progression and recurrence [3,4]. It is currently believed that CSCs are responsible for tumor initiation, progression and relapse, and that depletion of these cells is obligatory to cure patients

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