Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential

This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (SAF2008-03238) and Comunidad de Madrid (S-SAL/0185/2006) to the laboratory of J.M.A.The Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) is in part supported by an institutional grant from Fundación Ramón Areces.Peer reviewe

An Off-Target Nucleostemin RNAi Inhibits Growth in Human Glioblastoma-Derived Cancer Stem Cells

Glioblastomas (GBM) may contain a variable proportion of active cancer stem cells (CSCs) capable of self-renewal, of aggregating into CD133+ neurospheres, and to develop intracranial tumors that phenocopy the original ones. We hypothesized that nucleostemin may contribute to cancer stem cell biology as these cells share characteristics with normal stem cells. Here we report that nucleostemin is expressed in GBM-CSCs isolated from patient samples, and that its expression, conversely to what it has been described for ordinary stem cells, does not disappear when cells are differentiated. The significance of nucleostemin expression in CSCs was addressed by targeting the corresponding mRNA using lentivirally transduced short hairpin RNA (shRNA). In doing so, we found an off-target nucleostemin RNAi (shRNA22) that abolishes proliferation and induces apoptosis in GBM-CSCs. Furthermore, in the presence of shRNA22, GBM-CSCs failed to form neurospheres in vitro or grow on soft agar. When these cells are xenotransplanted into the brains of nude rats, tumor development is significantly delayed. Attempts were made to identify the primary target/s of shRNA22, suggesting a transcription factor involved in one of the MAP-kinases signaling-pathways or multiple targets. The use of this shRNA may contribute to develop new therapeutic approaches for this incurable type of brain tumor

Caracterización de células troncales tumorales derivadas de glioblastomas humanos; desarrollo de modelos preclínicos y ensayos terapéuticos

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-11-201

Viral targeting of glioblastoma stem cells with patient-specific genetic and post-translational p53 deregulations

Cancer therapy urges targeting of malignant subsets within self-renewing heterogeneous stem cell populations. We dissect the genetic and functional heterogeneity of human glioblastoma stem cells (GSCs) within patients by their innate responses to non-pathogenic mouse parvoviruses that are tightly restrained by cellular physiology. GSC neurospheres accumulate assembled capsids but restrict viral NS1 cytotoxic protein expression by an innate PKR/eIF2α-P response counteractable by electric pulses. NS1 triggers a comprehensive DNA damage response involving cell-cycle arrest, neurosphere disorganization, and bystander disruption of GSC-derived brain tumor architecture in rodent models. GSCs and cancer cell lines permissive to parvovirus genome replication require p53-Ser15 phosphorylation (Pp53S15). NS1 expression is enhanced by exogeneous Pp53S15 induction but repressed by wtp53. Consistently, patient-specific GSC subpopulations harboring p53 gain-of-function mutants and/or Pp53S15 are selective viral targets. This study provides a molecular foundation for personalized biosafe viral therapies against devastating glioblastoma and other cancers with deregulated p53 signaling.SAF2008-03238 and SAF2011-29403 (Spanish Ministerio de Ciencia e Innovacion), S2013/ABI-2906-FEDER (Comunidad de Madrid), and SAF2015-68522-P-MINECO/FEDER,UE (Spanish Ministerio de Economía y Competitividad and Ministerio de Ciencia, Investigación y Universidades) to J.M.A. The Centro de Biología Molecular Severo Ochoa (CSIC-UAM) is, in part, supported by institutional grants from Banco Santander and Fundación Ramón Areces

Efficacy of rapamycin against glioblastoma cancer stem cells

Purpose: The cancer stem cell (CSC) hypothesis suggests a hierarchical organization of cells within the tumor, in which only a subpopulation of stem-like cells is responsible for the rise and progression of the tumor. Glioblastomas (GBM), a lethal brain tumor, may contain a variable proportion of active CSCs. On the other hand, the phosphatidylinositol 3-kinase (PI3 K)/Akt/mammalian target of rapamycin (mTOR) pathway is highly active in up to 70 % of GBM. The kinase mTOR is a key component of the PI3K pathway that mediates the regulation of growth and cell survival signaling. However, clinical trials with rapamycin, an effective inhibitor of mTOR, have not been up to the created expectations and a plausible explanation is missing. In this work, we analyze the effect of rapamycin on the GBM-CSC population. Methods: The efficacy of rapamycin in vitro was tested on two primary cell lines derived from human GBM surgical resections that fulfill the criteria to be considered as CSCs. We confirmed the inhibition state of the PI3K/Akt/mTOR pathway analyzing the mTOR direct target ribosomal protein S6. We assayed the growth rate, CD133 expression and ability of forming colonies in soft agar of the CSCs under different doses of rapamycin. The efficacy of rapamycin in vivo was assayed in a CSCs-based orthotopic xenograft. Results and conclusions: We report the efficacy of rapamycin by reducing CSCs proliferation and tumorigenic potential in vitro. Despite these encouraging results, the efficacy in vivo was very poor. This finding confirms the limited use of rapamycin as a monotherapy for glioblastomas.Ministerio de Ciencia e Innovación (España)Ramón Areces FoundationDepto. de Estadística e Investigación OperativaFac. de FarmaciaTRUEpu

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential

This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (SAF2008-03238) and Comunidad de Madrid (S-SAL/0185/2006) to the laboratory of J.M.A.The Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM) is in part supported by an institutional grant from Fundación Ramón Areces.Peer reviewe

Glioma-Parvovirus Interactions: Molecular Insights and Therapeutic Potential

Brain tumours remain one of the most devastating diseases of modern medicine. Although they only represent approximately 1.9% of primary tumours in Europe, their mortality is around 70% and they are within the group of the 10 cancer types causing the highest yearly mortality rate. Gliomas are malignancies of neuroepithelial origin and represent 40-60% of brain tumours. In particular, glioblastoma multiforme (GBM, astrocytic tumours of type IV) is the most aggressive and frequent of primary brain tumours, representing 60% of gliomas. Despite clinical practice advances, the mean survival time of GBM patients has not improved significantly within the last few decades, and it remains around 12-15 months. Current standard of care includes maximal safe surgical resection, and a combination of radio- and chemotherapy with concomitant and adjuvant temozolomide or carmustine wafers (Wen and Kesari 2008). At the moment, the clinical improvement reached is modest, with a 5-year survival rate of less than 5% (Mangiola et al. 2010). The poor results obtained with conventional therapies may be explained by their relatively unspecific nature (Newton 2010), the inefficient delivery of many drugs to the tumoral tissue due to the blood-brain and blood-tumour barriers, as well as by the intrinsic radio- and chemo-resistance of GBM (Newton 2010). In light of the limitations of conventional treatment strategies, the necessity of new approaches that would be more effective against GBM became evident. The current understanding of the molecular biology of GBM has set researchers on the path of more targeted and specific therapies exploiting the molecular properties of the tumour. Most targeted agents are tyrosine kinase inhibitors, or monoclonal antibodies directed against either cell surface growth factor receptors or intercellular signaling molecules (angiogenesis) (Van Meir et al. 2010). The overall experience of the monotherapy with targeted agents has shown limited efficacy, with response rates of less than 10-15% and no prolongation of survival (Clarke et al. 2010; Van Meir et al. 2010). Other promising therapies for GBM are also currently being investigated, including combined therapy with targeted agents, immunotherapy, gene therapy, or oncolytic virotherapy (Clarke et al. 2010; Van Meir et al. 2010).Ministerio de Ciencia e Innovación (España)Comunidad de MadridCentro de Biología Molecular "Severo Ochoa"Fundación Ramón ArecesDepto. de Estadística e Investigación OperativaFac. de FarmaciaTRUEpu

Cell cycle regulation of the nuclear translocation of MVM capsid proteins.

<p><b>A</b>. MVM capsid proteins (VPs) are excluded from the nucleus at G0/G1. Microscopy analysis of mouse (MF-VPs) and human (HF-VPs) fibroblasts stably expressing VPs fixed as asynchronous cultures (async.), synchronized by density arrest (G1), or by isoleucine deprivation/aphidicolin (G1/S). <b>B</b>. Kinetic of VPs nuclear transport in quiescent (G0) mouse fibroblast induced into cycle by serum. <i>Left</i>, cells stained with the α-VPs and PCNA antibodies. <i>Right</i>, average percentages with standard errors from three experiments of VPs subcellular distribution at the indicated hps. <b>C</b>. VPs nuclear transport is allowed at S phase. PCNA and VPs subcellular distribution in synchronous MF-VPs at the indicated hpa. <b>D</b>. VPs phenotypes scored in mouse and human fibroblasts hours post-release (hpr) of synchronization by aphidicolin (G1/S), or by density arrest (G1). Bars represent average values with standard errors from three or four experiments. (NC (blue): predominantly nuclear).</p

Cell cycle dependence of VPs expression and capsid assembly in the synchronous MVM infection of mammalian fibroblasts.

<p>VPs expression and capsid formation in infected mouse (MFs) and human (HFs) fibroblasts analyzed by flow cytometry <b>(A, B)</b>, or IF <b>(C, D)</b>. Cells were synchronized by (<b>A</b>) isoleucine deprivation+aphidicolin (G1/S), or by (<b>B</b>) culturing to high density (G1), and stained for DNA content with DAPI (histograms: blank for mock, grey-filled for infected), VPs and Capsid (biparametric dot plots), at the indicated hours post-arrest releases (hpa or hpc, respectively). The percentage of cells in the corresponding gates is shown. CC, cell count. (<b>C</b>) Nuclear transport and assembly of MVM capsid subunits in synchronously infected mammalian fibroblasts. The figure shows representative fields of VPs and Capsid localized by double IF in MVM infected mouse and human fibroblats at the indicated hours post-release of the isoleucine/aphidicolin (G1/S) or growth to confluence (G1) cell cycle arrests. Scale bar 25 μm. <b>(D)</b> VPs subcellular distribution in synchronously infected mouse and human fibroblasts at the indicated hours post-release of the G1/S (hpa) or G1 (hpc) arrests. Average values with errors from four experiments are shown. (<b>E</b>) Synchronously infected MFs at G1/S stained for assembled capsids and viral DNA synthesis by FISH-hybridization (vDNA) along hpa. The figure shows representative confocal fields of cells from three experiments. Scale bar 10 μm.</p

Importins α/2β1 and transportin are not involved in VP2 nuclear import.

<p>Transport of VP2-trimer (t) analyzed by confocal microscopy in Digitonine-permeabilized HeLa cells, in combination with competitor markers. The transport phenotype of 200 ng of gradient-purified VP2(t) in the presence of 10 μg of BSA conjugated to peptides of characterized import routes (BSA-NLS and BSA-M9), or of 3.5 μM of NLS peptide alone (NLSp), are shown. Transport factors were provided by rabbit reticulocyte lysates (first five columns) or by purified importins (right columns). Representative results from three experiments are shown. Scale bar 10 μm.</p