Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Imp2 controls oxidative phosphorylation and is crucial for preserving glioblastoma cancer stem cells Michalina Janiszewska,1,8 Mario L. Suva`,2,8 Nicolo Riggi,2 Riekelt H. Houtkooper,3,4 Johan Auwerx,3 Virginie Cle´ment-Schatlo,5 Ivan Radovanovic,5 Esther Rheinbay,2,6 Paolo Provero,7 and Ivan Stamenkovic1,9 1Experimental Pathology, Department of Laboratories, CHUV, University of Lausanne, Lausanne CH-1011, Switzerland; 2James Homer Wright Pathology Laboratories, Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02114, USA; 3Laboratory for Integrative and Systems Physiology, Nestle Chair in Energy Metabolism (NCEM), Ecole Polytechnique Fe´de´rale de Lausanne, Lausanne CH-1015, Switzerland; 4Laboratory Genetic Metabolic Diseases, University of Amsterdam, Academic Medical Center, Amsterdam 1105 AZ, Netherlands; 5Department of Clinical Neurosciences, University Hospital of Geneva, Geneva CH-1211, Switzerland; 6Bioinformatics Program, Boston University, Boston, Massachusetts 02215, USA; 7Department of Genetics, Biology, and Biochemistry, University of Torino, Torino 10126 Italy Growth of numerous cancer types is believed to be driven by a subpopulation of poorly differentiated cells, often referred to as cancer stem cells (CSCs), that have the capacity for self-renewal, tumor initiation, and generation of nontumorigenic progeny. Despite their potentially key role in tumor establishment and maintenance, the energy requirements of these cells and the mechanisms that regulate their energy production are unknown. Here, we show that the oncofetal insulin-like growth factor 2 mRNA-binding protein 2 (IMP2, IGF2BP2) regulates oxidative phosphorylation (OXPHOS) in primary glioblastoma (GBM) sphere cultures (gliomaspheres), an established in vitro model for CSC expansion. We demonstrate that IMP2 binds several mRNAs that encode mitochondrial respiratory chain complex subunits and that it interacts with complex I (NADH:ubiquinone oxidoreductase) proteins. Depletion of IMP2 in gliomaspheres decreases their oxygen consumption rate and both complex I and complex IV activity that results in impaired clonogenicity in vitro and tumorigenicity in vivo. Importantly, inhibition of OXPHOS but not of glycolysis abolishes GBM cell clonogenicity. Our observations suggest that gliomaspheres depend on OXPHOS for their energy production and survival and that IMP2 expression provides a key mechanism to ensure OXPHOS maintenance by delivering respiratory chain subunit-encoding mRNAs to mitochondria and contributing to complex I and complex IV assembly. [Keywords: Imp2; cancer stem cells; OXPHOS; glioblastoma; respiratory complex; mitochondria] Supplemental material is available for this article. Received January 26, 2012; revised version accepted July 19, 2012. A growing number of malignancies are recognized to be the denomination of cancer stem cells (CSCs). Despite composed of phenotypically heterogeneous cells that being presumed to constitute the key cell subpopulation are hierarchically organized and have diverse degrees of that determines tumor development, CSCs are function- differentiating, proliferative, and tumorigenic capacity ally defined on the basis of no more than a handful of (Frank et al. 2010; Clevers 2011; Magee et al. 2012). The properties, chief among which are their ability to initiate apex of the hierarchy is believed to be occupied by un- tumor growth and give rise to all tumor cell subpopula- differentiated cells that can self-renew, give rise to rapidly tions in vivo as assessed by xenotransplantation assays. proliferating cell subpopulations, and reconstitute a phe- Resistance to therapy is often attributed to CSCs but does nocopy of the primary tumor upon injection into immu- not constitute a defining trait (Dean et al. 2005). A major nocompromised mice—properties that have earned them challenge to the isolation of CSCs from solid tumors is the paucity of reliable means to identify them. Whereas the cell surface receptors CD34 and CD38 have helped 8These authors contributed equally to the work. define leukemia stem cells based on their convincing 9Corresponding author validation as markers of normal hematopoietic stem cells E-mail [email protected] Article published online ahead of print. Article and publication date are (Lapidot et al. 1994), the developmental hierarchy of most online at http://www.genesdev.org/cgi/doi/10.1101/gad.188292.112. tissues that develop solid tumors is insufficiently char- 1926 GENES & DEVELOPMENT 26:1926–1944 Ó 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press Imp2 in glioblastoma cancer stem cells acterized to provide a robust basis for CSC identification. GBM based on their expression of diverse cell surface Several single cell surface receptors, including CD133, markers, including CD133 (Singh et al. 2004), SSEA1 (Son CD44, and SSEA, or combinations thereof, have been et al. 2009), and CD44 (Anido et al. 2010). Moreover, single used to identify CSCs in glioblastoma (GBM) based on self-renewing GBM cells form gliomaspheres in culture their expression in subpopulations of cells with self- that display properties consistent with CSC enrichment renewal and tumor-initiating properties (Singh et al. (Lee et al. 2006; Wakimoto et al. 2009, 2012). GBM thus 2004; Son et al. 2009; Anido et al. 2010). These receptors provides a suitable solid tumor model to investigate CSC have proved to be valid markers of CSCs in freshly re- properties that distinguish them from cells that comprise moved tumors, but it remains critical to complement the tumor bulk. We therefore addressed Imp2 expression, any marker used by stringent functional experiments to the repertoire of its target mRNAs, and its functional identify cells with high tumor-initiating potential whose relevance in GBM. Our observations show that Imp2 is study is essential for better understanding of tumor expressed in GBM and that its expression is highest in development. CD133+ cells from freshly isolated tumors, in gliomasphere Single cells with self-renewing and tumor-initiating cultures in vitro, and in cells associated with blood vessels capacity from a variety of solid tumors are able to form and necrotic areas in vivo, where CSCs are believed to spheres upon in vitro culture under defined serum-free reside. We also demonstrate that Imp2 binds mitochondrial conditions (Galli et al. 2004; Ricci-Vitiani et al. 2007). respiratory complex IV (CIV) mRNAs and complex I (CI) GBM spheres (gliomaspheres) have been shown to allow proteins and participates in their assembly and function, long-term expansion of cells with clonogenic and highly thereby regulating oxidative phosphorylation (OXPHOS). tumorigenic properties that recapitulate the morphology Remarkably, Imp2 expression is shown not only to main- and genetic mutations of the parental tumor (Lee et al. tain OXPHOS in tumor cells, but to be required for the 2006; Wakimoto et al. 2012). They therefore appear to be survival and function of cells that display self-renewal and enriched in cells that correspond to the current func- tumor-initiating properties. Our observations define a new tional definition of CSCs and represent a reproducible function for Imp2 that may constitute a therapeutic target functional model to address CSC properties. in one of the most malignant human tumors. Because CSCs may, in some cases, display plasticity that is typically associated with developing tissues, onco- fetal proteins may participate in and potentially even Results determine many of their phenotypic and functional fea- Imp2 expression in normal brain and glial tumors tures. The oncofetal protein Imp2 (insulin-like growth factor 2 mRNA-binding protein 2) is expressed in the Immunohistochemical staining of a normal adult brain developing mammalian brain, suggesting possible impli- and a panel of gliomas of varying grades revealed that Imp2 cation in normal embryonic development (Christiansen is absent in normal brains and grade II and III astrocytomas et al. 2009). Imp2 belongs to the Imp family of proteins (Fig. 1A), whereas 40 out of 51 GBM samples were Imp2- that regulate subcellular mRNA localization, translation, positive. Correlation between Imp2 expression and glioma and stability (Christiansen et al. 2009) and consists of grading was confirmed by microarray analysis of Imp2 three pairs of RNA-binding domains: RRM1–2, KH1–2, expression in an independent data set of 153 gliomas of and KH3–4. Studies on paralog proteins IMP1 and IMP3 different grades and 23 normal samples (Fig. 1B; Sun et al. have shown a correlation between their expression and 2006). Some variability in Imp2 expression is observed poor outcome in various malignancies, including mela- among GBM subtypes, as defined according to The Cancer noma, ovarian cancer, and pancreatic, prostate, thyroid, Genome Atlas (TCGA) database (http://cancergenome. breast, and colon carcinoma (Yaniv and Yisraeli 2002; nih.gov), with the lowest levels found in the neural sub- Himoto et al. 2005a,b; Dimitriadis et al. 2007; Kobel et al. type (Fig. 1C). Interestingly, elevated Imp2 expression 2007), but mechanisms that underlie their functional correlates with poor prognosis in the proneural GBM implication in tumor biology have not been identified subtype (Fig. 1D),