An Off-Target Nucleostemin RNAi Inhibits Growth in Human Glioblastoma-Derived Cancer Stem Cells Jon Gil-Ranedo 1 , Marina Mendiburu-Elic ¸abe 1 , Mercedes Garcı´a-Villanueva 2 , Diego Medina 3 , Marta del A ´ lamo 3 , Marta Izquierdo 1 * 1 Departamento de Biologı ´a Molecular, Universidad Auto ´ noma de Madrid, Centro de Biologı ´a Molecular Severo Ochoa, Consejo Superior de Investigaciones Cientı ´ficas, Instituto Sanitario Ramo ´ n y Cajal, Madrid, Spain, 2 Servicio de Anatomı ´a Patolo ´ gica, Hospital Universitario Ramo ´ n y Cajal, Madrid, Spain, 3 Servicio de Neurocirugı ´a, Hospital Universitario Ramo ´ n y Cajal, Instituto Sanitario Ramo ´ n y Cajal, Madrid, Spain Abstract 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. Citation: Gil-Ranedo J, Mendiburu-Elic ¸abe M, Garcı ´a-Villanueva M, Medina D, del A ´ lamo M, et al. (2011) An Off-Target Nucleostemin RNAi Inhibits Growth in Human Glioblastoma-Derived Cancer Stem Cells. PLoS ONE 6(12): e28753. doi:10.1371/journal.pone.0028753 Editor: Keith L. Black, Cedars-Sinai Medical Center, United States of America Received June 28, 2011; Accepted November 14, 2011; Published December 12, 2011 Copyright: ß 2011 Gil-Ranedo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by funds from: Ministerio de Ciencia e Innovacio ´ n (SAF 2009-07259) Spain, and Fundacio ´ n Eugenio Rodriguez Pascual (Madrid). The Centro de Biologı ´a Molecular S.O. is also the recipient of an institutional grant from the Ramo ´ n Areces Foundation. JG-R was the recipient of a predoctoral fellowship from the Gobierno Vasco, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: mizquierdo@cbm.uam.es Introduction Glioblastoma multiforme is one of the most malignant and common of all astrocytic tumors [1]. The growth pattern of GBM is highly infiltrative, rendering a surgical cure very difficult and resulting in very poor survival outcomes that have improved only marginally in the past several decades [2]. The cancer stem cell hypothesis suggests that tumors are organized in a hierarchy with a subpopulation of CSCs responsible for tumor progression, maintenance, and recurrence [3]. Cells with stem-like properties were initially identified in acute myeloid leukaemia [4], and at present their existence has been confirmed in breast cancer [5], medulloblastoma and glioblastoma [6], prostate cancer [7], melanoma [8], ovarian cancer [9], head and neck squamous carcinomas [10], colon cancer [11], pancreatic cancer [12] and lung cancer [13], among others. In glioblastoma, relapses normally follow treatment, probably because CSCs are highly infiltrative, selectively resistant to radiotherapies, chemotherapies [14,15,16], immunotherapies [17], and promote angiogenic activity. More- over, chemo- and radio- therapies may prime brain tumor CSCs to enhance their stem-cell-like characteristics [18]. This population of CSCs is highly tumorigenic and phenocopy the original tumor in rodent xenograft models [19,20]. Approaches to force CSCs to differentiate to cells with limited, or no cell division attributes, by exposing them to bone morphogenetic proteins for example, were used to render them more vulnerable to conventional therapies, and showed considerable efficacy in mouse models [21]. Understanding the basic biology of cancer stem cells is a key feature before moving into putative treatments to eliminate them. Nucleostemin is a GTP-binding protein, so called because of its nucleolar localization and preferential expression in stem cells [22]. Although the protein is predominantly present in embryonic and adult stem cells, it is also expressed in several transformed cell lines and tumors [23,24]. Nucleostemin, on the other hand, is abruptly down-regulated during differentiation prior to terminal cell division. This protein was first identified in adult rat neural stem cells, and has been implicated in cell-cycle progression [22]. Several nucleostemin-binding proteins have been identified, including p53, MDM2 and the telomeric repeat binding factor 1 (TRF1) [22,25,26]. Alterations in nucleostemin expression levels cause a decrease in the proliferation rate of cells, in both p53 dependent and independent manners, [22,26–28]. The protein is indispensable for early embryogenesis [29] but is also important in adult neural stem cells [30]. Some studies have shown that depletion of nucleostemin is associated with a limited tumorigenic capacity in both HeLa and PC-3 cells [31,24]. Beside its implication in the regulation of cell proliferation, several additional roles have been assigned to nucleostemin such as telomere length PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28753