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Rnai Screening in Glioma Stem-Like Cells Identifies PFKFB4 As a Key

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Rnai Screening in Glioma Stem-Like Cells Identifies PFKFB4 As a Key Oncogene (2012) 31, 3235–3243 & 2012 Macmillan Publishers Limited All rights reserved 0950-9232/12 www.nature.com/onc ORIGINAL ARTICLE RNAi screening in glioma stem-like cells identifies PFKFB4 as a key molecule important for cancer cell survival V Goidts1,2, J Bageritz1, L Puccio1, S Nakata1, M Zapatka1, S Barbus1, G Toedt1, B Campos3, A Korshunov4,5, S Momma6, E Van Schaftingen7, G Reifenberger8, C Herold-Mende3, P Lichter1 and B Radlwimmer1 1Division of Molecular Genetics, German Cancer Research Center, Heidelberg, Germany; 2Department of Pediatric Oncology, Hematology and Immunology, Children’s Hospital, University of Heidelberg, Heidelberg, Germany; 3Division of Neurosurgical Research, Department of Neurosurgery, University of Heidelberg, Heidelberg, Germany; 4Department of Neuropathology, University of Heidelberg, Heidelberg, Germany; 5Clinical Cooperation Unit Neuropathology, German Cancer Research Center, Heidelberg, Germany; 6Edinger Institute, Frankfurt University Medical School, Frankfurt, Germany; 7Laboratory of Physiological Chemistry, de Duve Institute, Universite´ Catholique de Louvain, Brussels, Belgium and 8Department of Neuropathology, Heinrich-Heine-University, Du¨sseldorf, Germany The concept of cancer stem-like cells (CSCs) has gained Introduction considerable attention in various solid tumors including glioblastoma, the most common primary brain tumor. Glioblastoma is the most common and most malignant This sub-population of tumor cells has been intensively primary brain tumor in adults. Despite multimodal investigated and their role in therapy resistance as well as aggressive treatment, comprising surgical resection, tumor recurrence has been demonstrated. In that respect, local radiation therapy and systemic chemotherapy, development of therapeutic strategies that target CSCs the average patient survival time is still in the range of 1 (and possibly also the tumor bulk) appears a promising year after diagnosis (Stupp et al., 2007). Several recent approach in patients suffering from primary brain tumors. studies suggested a hierarchical cellular organization in In the present study, we utilized RNA interference (RNAi) glioblastomas, which consist of heterogeneous cell to screen the complete human kinome and phosphatome populations that differ in their tumorigenic potential. (682 and 180 targets, respectively) in order to identify Indeed, it is believed that gliomas are initiated and genes and pathways relevant for the survival of brain maintained by a small sub-population of cells that is CSCs and thereby potential therapeutical targets for capable of extensive self-renewal and recapitulation of glioblastoma. We report of 46 putative candidates the original tumor in nonobese diabetic/severe com- including known survival-related kinases and phospha- bined immunodeficient mice (Singh et al., 2003; Lee tases. Interestingly, a number of genes identified are et al., 2006; Vescovi et al., 2006). These so-called brain involved in metabolism, especially glycolysis, such as cancer stem-like cells (CSCs) have been implicated not PDK1 and PKM2 and, most prominently PFKFB4. only in tumor initiation, but also in therapy resistance In vitro studies confirmed an essential role of PFKFB4 and tumor recurrence. It was shown that brain CSCs are in the maintenance of brain CSCs. Furthermore, high enriched in recurrent malignant gliomas and are PFKFB4 expression was associated with shorter survival resistant to chemo- and radiotherapy (Bao et al., 2006; of primary glioblastoma patients. Our findings support the Eramo et al., 2006; Liu et al., 2006; Al-Hajj, 2007). importance of the glycolytic pathway in the maintenance Given their central role, it has appeared that of malignant glioma cells and brain CSCs and imply therapeutically targeting this particular population of tumor metabolism as a promising therapeutic target in cells is crucial for successful glioblastoma treatment glioblastoma. (Reya et al., 2001; Pardal et al., 2003; Park et al., 2009). Oncogene (2012) 31, 3235–3243; doi:10.1038/onc.2011.490; One possible direction is to uncover the mechanisms, by published online 7 November 2011 which brain CSCs escape apoptosis, and ultimately provide knowledge for the development of drug Keywords: loss-of-function screen; apoptosis; glioblas- therapeutics targeting the involved genes and pathways. toma; cancer stem-like cells; glycolysis Kinases and phosphatases control the reversible process of phosphorylation, which is involved in each intracellular pathway, including cell survival signalling. Therefore, identification of the kinases and phospha- tases whose inhibition induces the death of brain CSCs Correspondence: Dr V Goidts, Division of Molecular Genetics, may pave the way toward novel therapeutic targets. In German Cancer Research Center, Im Neuenheimer Feld, 280, 69120 this study, we performed a loss-of-function screen using Heidelberg, Germany. E-mail: [email protected] a lentiviral short-hairpin RNA (shRNA) library repre- Received 17 November 2010; revised 12 September 2011; accepted 15 senting the entire human kinome and phosphatome, and September 2011; published online 7 November 2011 identified 46 candidates that are essential for the survival Brain cancer stem-like cells require PFKFB4 for survival V Goidts et al 3236 2.5 of brain CSCs in vitro. These candidates included key BMPR2 NTRK1 PAK2 PRKCA regulators of the glycolytic pathway, underlining the 2.0 central role of metabolism for the survival of brain CSCs. One of the glycolysis-associated candidate genes, 1.5 PFKFB4, was functionally characterized in brain CSCs 1.0 in vitro, and the importance of lactate and adenosine 0.5 triphosphate (ATP) production for the survival of brain (relative to scrambled shRNA) 0.0 CSCs was assessed. Fold change of PI-positive cells Kinases and phosphatases Figure 1 Identification of survival kinases and phosphatases. NCH421k cells were seeded in 96-well plates and transduced with Results shRNAs directed against all known and putative kinases and phosphatases in the human genome. Cells were incubated for 6 days to allow target knockdown and cell death was measured Kinome and phosphatome RNA interference (RNAi) by PI staining and analyzed by FACS. The graph depicts the screen to induce brain CSC death median fold change of cell death for all kinases and phosphatases Lentiviral short-hairpin libraries were used recently to normalized to control shRNA. The genes whose loss of function conduct loss-of-function screens in mammalian cell lines increased cell death by 41.5-fold were considered as positive hits. The red line represents the median of all shRNAs plus 2.5-fold and turned out to be particularly useful in slowly median absolute deviation (MAD). dividing cells (Wurdak et al., 2010). We performed a large-scale silencing screen in vitro to identify kinases and phosphatases that are essential for the survival of Table 1 Putative survival kinases identified in the RNA interference brain CSCs. We transduced NCH421k, whose stem-like (RNAi) screen cell properties have been previously characterized (Campos et al., 2010), with a lentiviral vector-based Symbol Accession number Cell death fold change shRNA library that targets each of the 682 and 180 PAK2 NM_002577 2.08 known and putative kinases and phosphatases, respec- PRKCA NM_002737 2.05 tively (average of 4.5 shRNAs per target, The RNAi NTRK1 NM_002529 2.05 Consortium, Broad Institute from MIT and Harvard, BMPR2 NM_001204 2.02 MGC75495 XM_292160 1.85 MA). Viral stocks were produced in a 96-well plate TTBK2 NM_173500 1.85 format and used at a sufficient titer to transduce brain PRKCI NM_002740 1.85 CSCs. PFKFB4 NM_004567 1.82 In order to assess the efficiency of the viral transduc- ETNK2 NM_018208 1.79 tion, cells transduced with lentiviral particles produced EPHA5 NM_004439 1.77 RIPK1 NM_003804 1.73 from a control plasmid encoding green fluorescent SBK1 XM_370948 1.72 protein (GFP) were included in each plate. Viral PKM2 NM_182471 1.72 transduction was efficient as indicated by 70–90% ITK NM_005546 1.72 GFP-expressing cells. As positive control for the cell MAPKAPK5 NM_003668 1.69 PRKX NM_005044 1.68 death phenotype, brain CSCs were transduced with a NME3 NM_002513 1.68 specific shRNA targeting BCL2. After 6 days, cell death PIP5K1A NM_003557 1.68 was measured by propidium iodide staining and FLT1 NM_002019 1.65 fluorescence-activated cell sorting (FACS). In the BRD2 NM_005104 1.65 screen, we considered only those genes as positive hits CABC1 NM_020247 1.64 FN3KRP NM_024619 1.63 whose median over all shRNAs targeting this gene LCK NM_005356 1.62 showed an increased level of cell death of X1.5-fold MAP4K4 NM_145687 1.61 (median of all shRNAs plus 2.5-fold median absolute RIOK2 NM_018343 1.59 deviation) relative to that of the control (scrambled) ITPKC NM_025194 1.57 SPHK1 NM_182965 1.57 shRNA. Overall, 39 kinases and 7 phosphatases fulfilled MAP3K1 XM_042066 1.56 this criterion (Figure 1 and Tables 1 and 2) and were PDK1 NM_002610 1.55 defined as survival genes. Moreover, each of these genes SPHK2 NM_020126 1.55 was targeted by at least two shRNAs that showed a fold SGK NM_005627 1.54 increase of cell death rate X1.5-fold. STK25 NM_006374 1.54 CHEK2 NM_007194 1.53 ADRBK1 NM_001619 1.52 Validation of survival genes BMPR1B NM_001203 1.51 LIMK2 NM_016733 1.51 Next, we generated a focused library comprising the top STK32C NM_173575 1.51 10 candidates from the first screen, each represented by TP53RK NM_033550 1.50 2 to 3 independent shRNAs that gave the highest rate of TAOK1 NM_020791 1.50 cell death. To ensure that the differences observed were not caused by ‘off-target’ shRNA effects, we verified the level of silencing of each shRNA by quantitative real- independent shRNAs that decreased their target mRNA time reverse transcription–PCR (qRT–PCR). Of these transcript levels by 2- to 10-fold, suggesting that the top 10 genes, 9 were represented by at least two observed phenotype was because of the silencing of the Oncogene Brain cancer stem-like cells require PFKFB4 for survival V Goidts et al 3237 Table 2 Putative survival phosphatases identified in the RNA S2) showed an increased fold change of apoptosis in all interference (RNAi) screen cell lines.
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