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

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