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• W2890593886 abstract "Scientific Report13 September 2018free access Source DataTransparent process SIRT2-mediated inactivation of p73 is required for glioblastoma tumorigenicity Kosuke Funato Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Department of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Tomoatsu Hayashi Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Kanae Echizen Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Lumi Negishi Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Naomi Shimizu Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Ryo Koyama-Nasu Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yukiko Nasu-Nishimura Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yasuyuki Morishita Department of Human Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Viviane Tabar Department of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Tomoki Todo Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yasushi Ino Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Akitake Mukasa Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Nobuhito Saito Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Tetsu Akiyama Corresponding Author [email protected] orcid.org/0000-0002-4215-5073 Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Kosuke Funato Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Department of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Tomoatsu Hayashi Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Kanae Echizen Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Lumi Negishi Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Naomi Shimizu Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Ryo Koyama-Nasu Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yukiko Nasu-Nishimura Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yasuyuki Morishita Department of Human Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Viviane Tabar Department of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA Search for more papers by this author Tomoki Todo Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Yasushi Ino Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Akitake Mukasa Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Nobuhito Saito Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Tetsu Akiyama Corresponding Author [email protected] orcid.org/0000-0002-4215-5073 Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Author Information Kosuke Funato1,2,‡, Tomoatsu Hayashi1,‡, Kanae Echizen1, Lumi Negishi1, Naomi Shimizu1, Ryo Koyama-Nasu1, Yukiko Nasu-Nishimura1, Yasuyuki Morishita3, Viviane Tabar2, Tomoki Todo4, Yasushi Ino4, Akitake Mukasa4, Nobuhito Saito4 and Tetsu Akiyama *,1 1Laboratory of Molecular and Genetic Information, Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 2Department of Neurosurgery, Memorial Sloan-Kettering Cancer Center, New York, NY, USA 3Department of Human Molecular Pathology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan 4Department of Neurosurgery, Faculty of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 3 5841 7834; Fax: +81 3 5841 8482; E-mail: [email protected] EMBO Rep (2018)19:e45587https://doi.org/10.15252/embr.201745587 PDFDownload PDF of article text and main figures.AM PDF Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Glioblastoma is one of the most aggressive forms of cancers and has a poor prognosis. Genomewide analyses have revealed that a set of core signaling pathways, the p53, RB, and RTK pathways, are commonly deregulated in glioblastomas. However, the molecular mechanisms underlying the tumorigenicity of glioblastoma are not fully understood. Here, we show that the lysine deacetylase SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cells, including glioblastoma stem cells. Furthermore, we demonstrate that SIRT2 regulates p73 transcriptional activity by deacetylation of its C-terminal lysine residues. Our results suggest that SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells and that SIRT2 may be a promising molecular target for the therapy of glioblastoma. Synopsis SIRT2 targets the tumor suppressor p73, thereby deacetylating its C-terminus and suppressing its transcriptional activity. SIRT2-mediated inactivation of p73 is crucial for the tumorigenicity of glioblastoma cells. The lysine deacetylase SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cells. SIRT2 targets and regulates the transcriptional activity of the tumor suppressor p73. SIRT2-mediated inactivation of p73 is critical for the proliferation and tumorigenicity of glioblastoma cells. Introduction Glioblastoma is the most malignant form of glioma. Despite great efforts, the median survival of glioblastoma patients has remained at around 1 year for the past decade 1. Several hallmark features of glioblastoma contribute to its aggressive phenotypes, such as uncontrolled proliferation, diffuse infiltration into surrounding brain tissue, and poor response to therapeutic agents. Furthermore, it has been reported that glioblastoma stem cells (GSCs), subsets of glioblastoma cells that possess the capability of self-renewal and exhibit extensive tumorigenicity, are resistant to both chemotherapy and radiotherapy and thus are responsible for the poor prognosis of glioblastoma 2, 3. Integrative genomic analyses have revealed that the p53, RB, and RTK pathways, termed core signaling pathways, are commonly deregulated in glioblastoma 2, 4, 5. Genetic alterations in these pathways enable glioblastoma cells to escape from cell-cycle checkpoints, apoptosis, and senescence, resulting in uncontrolled proliferation and enhanced survival. It has also been reported that mutations and deletions in neurofibromatosis type 1 (NF1) gene occur in 23% of glioblastoma 5. Furthermore, many glioblastomas have a heterozygous deletion of the NF-κB inhibitor α (NFKB1A) gene, which encodes IκB, a negative regulator in the NF-κB pathway 6, and/or mutations in the isocitrate dehydrogenase 1 (IDH1) gene 4. Sirtuins [class III (NAD-dependent) histone deacetylases (HDACs)] are conserved from bacteria to human and have been implicated in aging and longevity, resulting from their regulation of genomic stability and metabolism 7. There are seven sirtuin members in mammals, sirtuin 1–7 (SIRT1–7), and these exhibit diverse functions, including transcriptional silencing, metabolic regulation, and apoptosis. SIRT2 is localized to both the nucleus and the cytoplasm 8-11 and deacetylates α-tubulin, FOXO1, FOXO3a, p300, Lys16 of histone H4, Slug, CDH1, CDC20, and p53 8-13. SIRT2 also deacetylates the transcriptional repressor Slug to prevent its degradation and thereby controls malignancy of basal-like breast cancer 11. Furthermore, a SIRT2-specific inhibitor, a thiomyristoyl lysine compound, promotes c-Myc ubiquitination and degradation and exhibits broad anticancer activity 14. It has also been reported that SIRT2 regulates cell-cycle progression and genome stability by modulating the mitotic deposition of H4K20 methylation 15. In addition, it has been reported that SIRT inhibitors induce cell death and p53 acetylation through targeting both SIRT1 and SIRT2 13. In this study, we show that SIRT2 is required for the proliferation and tumorigenicity of glioblastoma cell. We further demonstrate that the suppression of glioblastoma proliferation by knockdown of SIRT2 requires p73. We find that SIRT2 regulates the transcriptional activity of p73 by deacetylation of its C-terminal lysine residues. Our results suggest that SIRT2 regulates the survival of glioblastoma via repression of p73 function. Results and Discussion Knockdown of SIRT2 induces the growth arrest and apoptosis of glioblastoma cells Using cells isolated from human glioblastoma, we have established a series of glioblastoma neurospheres (GB1–GB13 and GB16). We identified loss-of-function mutations in the DNA-binding domain of p53 in two of these glioblastoma neurospheres, GB2 and GB16, which result in Ser241 and His193 being replaced with Phe and Arg, respectively (Appendix Table S1). In addition, we identified EGFR amplification in GB13. Moreover, we previously showed that GB2 possesses the capability of self-renewal and exhibits extensive tumorigenicity 16. To identify novel therapeutic targets for glioblastoma cells, we performed an RNA interference (RNAi) screen using GB2, which is easy to culture and possesses high tumorigenic activity. GB2 cells were transduced with an siRNA library targeting 246 genes commonly expressed in glioblastoma neurospheres (Appendix Table S2) and then assayed for CD133 expression by quantitative RT–PCR (qRT–PCR) (Appendix Fig S1). CD133 has been successfully used as a stem cell marker for some glioblastomas 3, 17, 18, and it was previously shown that CD133 can be used as a stem cell marker for the glioblastoma spheres which were derived from the same cell specimen as GB2 19. Candidate genes that modulated CD133 expression more than twofold (Appendix Fig S1 and Appendix Table S2) were further validated for their effects on CD133 and/or nestin expression. From this screen, we identified SIRT2 as a candidate modulator of these properties of GB cells (Figs 1A and EV1A, Appendix Fig S1, Appendix Tables S2 and S3). In these experiments, knockdown of SIRT2 led to an increase in the acetylation of α-tubulin, a known substrate of SIRT2 9, indicating that SIRT2 was functionally suppressed in these cells (Fig EV1B). We also found that knockdown of SIRT2 resulted in significant inhibition of sphere formation in other primary glioblastoma neurospheres (GB4, GB11, GB13, and GB16) and glioblastoma cells isolated freshly from tumor samples (GB15) (Fig EV1A and B). Furthermore, limiting dilution assays confirmed that knockdown of SIRT2 caused inhibition of primary glioblastoma sphere formation (GB16) (Figs 1B and EV1C). In addition, we examined the effects of eight out of the top 10 candidate genes on the expression of Sox2, EZH2, and Olig2. We found that EHMT1, PTPRO, PTCH1, and TAL1 as well as SIRT2 suppressed the expression of Sox2, EZH2, and Olig2 (Appendix Table S3). Figure 1. Knockdown of SIRT2 using siRNA or treatment with AGK2 induces growth arrest and apoptosis of glioblastoma cells Sphere formation of GB2 cells transfected with an shRNA targeting SIRT2 was analyzed by an In Cell Analyzer 2000. Primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± SD of 10 wells. Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16. The figure shows a representative result of three independent experiments. mRNA levels of the indicated genes in GB2, GB4, and GB16 cells infected with a lentivirus expressing an shRNA targeting SIRT2 were measured by qRT–PCR. The results were normalized with the values for GAPDH. Bars indicate mean ± SD (n = 3–4). The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. GB17 was infected with a control (Empty) or shSIRT2-expressing (shS2 #1) lentivirus. Bars indicate mean ± SD of eight wells. The sphere formation capacity of CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample. (Left panel) GB18 was treated with AGK2 (10 μM) or DMSO. (Right panel) Secondary sphere formation of GB18 was examined in the absence of AGK2. Bars indicate mean ± SD of eight wells. Data information: Statistical significance was evaluated using the likelihood ratio test (for panel B) or unpaired two-tailed t-test. *P < 0.05; **P < 0.01. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Knockdown of SIRT2 induces growth arrest in glioblastoma cells (related to Fig 1) Sphere-forming capacities of GB cells transfected with an shRNA targeting SIRT2. GB cells were plated on 96-well plates at the indicated cell numbers. After 10 days of incubation, the spheres were analyzed by microscopy or using an In Cell Analyzer 2000. For GB15 and GB16, primary spheres were re-plated to evaluate secondary sphere formation. Bars indicate mean ± SD of 10 wells. GB cells infected with a lentivirus expressing an shRNA targeting SIRT2 were subjected to immunoblotting analysis using the indicated antibodies. Knockdown of SIRT2 causes a decrease in the sphere formation capacity of GB16. Estimated stem cell frequencies were determined from the data shown in Fig 1B by extreme limiting dilution analysis (http://bioinf.wehi.edu.au/software/elda). GB cells transfected with a lentivirus expressing an shRNA targeting SIRT2 were plated on laminin-coated 12-well plates (1 × 105 cells per well). After 5 days of incubation, the number of viable cells was counted at the indicated time points by Trypan blue staining. Bars indicate mean ± SD (n = 3). GB2 cells were infected with a lentivirus expressing an shRNA targeting SIRT2 and plated in 96-well plates. After 12 days of incubation under the non-adherent condition, the number of viable cells was counted. Bars indicate mean ± SD (n = 3). GB2 cells cultured in serum-free or serum-containing medium for more than 10 passages were infected with a control or shSIRT2-expressing lentivirus. After 11 days, cells were analyzed for sub-G0 DNA content. Bars indicate mean ± SD (n = 3). Knockdown of SIRT2 causes an increase in the levels of PUMA expression and cleavage of caspase-3 in GB2 cells cultured in the presence or absence of serum. Five days after viral infection, cells were subjected to qRT–PCR analysis for SIRT2 expression (left panel) and to immunoblotting analysis using the indicated antibodies (right panel). Bars indicate mean ± SD (n = 3). Data information: Statistical significance was evaluated using unpaired two-tailed t-test. *P < 0.05; **P < 0.01; N.S., not significant. Source data are available online for this figure. Download figure Download PowerPoint We also found that knockdown of SIRT2 significantly decreased the number of viable GB2, GB4, and GB16 cells (Fig EV1D and E). Furthermore, SIRT2 knockdown in GB2 cells resulted in an increase in the sub-G0 DNA fraction and an increase in the cleavage of caspase-3, indicating that SIRT2 knockdown induces apoptosis of GB2 cells (Fig EV1F and G). Consistent with these results, knockdown of SIRT2 resulted in an increased expression of multiple apoptotic and cell-cycle-regulating genes, such as PUMA, NOXA, and GADD45 in GB2, GB4, and GB16 cells (Figs 1C and EV1G). It is known that glioblastoma cells lose many of their original properties when cultured in serum-containing medium, including their stem cell-like characteristics 19. In contrast to GB2 cells cultured in serum-free medium, knockdown of SIRT2 in GB2 cells cultured in serum-containing medium barely caused increases in the sub-G0 DNA fraction (Fig EV1F) and the level of PUMA expression or cleavage of caspase-3 (Fig EV1G). Of particular note, the expression of SIRT2 was significantly decreased in GB2 cells cultured in serum-containing medium. It has been reported that GSCs can be enriched using several cell-surface markers, including CD133 3, 17-20. To further investigate the role of SIRT2 in GSCs, we sorted CD133-positive cells by FACS from GB17 and GB18 samples. Consistent with previous reports 19, CD133-positive cells had higher sphere formation capacity than CD133-negative cells (Fig 1D and E). We found that knockdown of SIRT2 resulted in the suppression of sphere formation of CD133-positive cells (Fig 1D). These results suggest that SIRT2 is required for the growth of GSCs. The SIRT2-specific inhibitor AGK2 induces growth arrest and apoptosis of glioblastoma cells AGK2 is a SIRT2-specific inhibitor that holds promise as an agent for the treatment of Parkinson's and other neurodegenerative diseases 21, 22. When GB2, GB4, GB11, or GB16 cells were treated with AGK2, their sphere formation capacities were reduced compared to vehicle-treated cells (Fig EV2A and B). Treatment with AGK2 caused an increase in α-tubulin acetylation, indicating that SIRT2 deacetylase activity was functionally inhibited (Fig EV2C). In addition, the reduction in GB2 cell number caused by lentivirus expression of an shRNA against SIRT2 was not further reduced by AGK2 treatment, suggesting that the effect of AGK2 is specific to SIRT2 (Fig EV2D). Furthermore, AGK2 treatment suppressed sphere formation of CD133-positive cells freshly isolated from a patient sample (GB18) (Figs 1E and EV2E). Click here to expand this figure. Figure EV2. The SIRT2-specific inhibitor AGK2 induces growth arrest of glioblastoma cells (related to Fig 1) GB2 cells cultured in the presence of AGK2 at the indicated concentration or DMSO were subjected to sphere formation assays. Bars indicate mean ± SD of 10 wells. Treatment of GB glioblastoma neurospheres with SIRT2 inhibitors, but not with EX527, causes a decrease in the number of spheres. Cells were plated in 96-well cell culture plates at the indicated cell numbers. After incubation with the indicated inhibitors for 8 days, the number of spheres was counted under a microscope. Bars indicate mean ± SD of at least 8 wells. GB2 cells were treated with 20 μM AGK2 or DMSO for 6 h and subjected to immunoblotting analysis using the indicated antibodies. AGK2 treatment is not additive with SIRT2 knockdown in suppressing GB2 cell proliferation. Cells infected with a control or shSIRT2-expressing (shS2 #1) lentivirus were treated with AGK2 (10 μM) or DMSO for 24 h. The number of viable cells was counted by Trypan blue staining. Bars indicate mean ± SD of triplicate technical repeats (n = 2). CD133-positive and CD133-negative cells sorted by FACS directly from a tumor sample (GB18) were subjected to sphere formation assays in the presence of AGK2 (10 μM) or DMSO. Scale bar, 200 μm. Treatment of GB2 cells with AGK2 increases the expression of PUMA, BAX, NOXA, and GADD45, but decreases the expression of CD133. GB2 cells (1.5 × 105 cells) were treated with 20 μM AGK2 or DMSO for 72 h and subjected to qRT–PCR to determine the mRNA expression levels of the indicated genes. Bars indicate mean ± SD (n = 3–4). p53 mutant GB cells do not respond to treatment with doxorubicin or camptothecin. 2 × 104 cells were treated with 20 μM AGK2, 5 μM doxorubicin (DXR), 1 μM camptothecin (CPT), or DMSO (control), respectively. After 4 h of incubation, the mRNA expression of the indicated genes was measured by qRT–PCR. Bars indicate mean ± SD (n = 3). AGK2 treatment induces apoptosis of GB2 cells. GB2 cells (5 × 105 cells) were treated with 20 μM AGK2 or DMSO for the indicated times and subjected to sub-G0 assays (n = 3). Treatment of GB2 cells with AGK2 increases the amount of cleaved caspase-3. GB2 cells (1.5 × 105 cells) were treated with 20 μM AGK2 or DMSO for the indicated times and subjected to immunoblotting with anti-caspase antibody. GB2 cells were treated with AGK2 as indicated, and caspase-3/7 activation was measured using the caspase-3/7 Glo assay. Bars indicate mean ± SD (n = 4). Data information: Statistical significance was evaluated using unpaired two-tailed t-test. *P < 0.05; **P < 0.01. Source data are available online for this figure. Download figure Download PowerPoint Consistent with the results of SIRT2 knockdown experiments, AGK2 treatment increased the expression of the p53-inducible genes, PUMA, NOXA, and GADD45, in GB2 cells (Fig EV2F). By contrast, treatment with camptothecin or doxorubicin failed to induce p53 target genes (Fig EV2G). Furthermore, we detected an increase in the sub-G0 DNA fraction and increased cleavage of caspase-3 in AGK2-treated GB2 cells (Fig EV2H and I). We also measured caspase-3/7 activation using the caspase-3/7 Glo assay and confirmed these results (Fig EV2J). Thus, AGK2 treatment induces apoptosis of GB2 cells. Taken together, these findings suggest that the deacetylase activity of SIRT2 is required for the proliferation and survival of glioblastoma cells. Among the family of sirtuins, SIRT1 is known to be involved in the development of cancer by blocking senescence and apoptosis 7. It has also been reported that SIRT1 inhibition enhances radiosensitivity of CD133-positive glioblastoma cells 23. We therefore examined the effects of SIRT1 inhibition on the proliferation and survival of glioblastoma cells. Salermide is an inhibitor of both SIRT1 and SIRT2 24, while EX527 is a specific inhibitor of SIRT1 25. Similar to AGK2, salermide treatment decreased the sphere formation and proliferation of GB2, GB4, GB11, and GB16 cells and induced the expression of PUMA and GADD45 in GB2 cells (Figs EV2B and EV3A and B). By contrast, EX527 barely suppressed proliferation and sphere formation even at a concentration (20 μM) 500 times higher than its IC50 (38 nM) (Figs EV2B and EV3B–D). EX527 treatment did not induce the expression of PUMA or GADD45 (Fig EV3E). Furthermore, knockdown of SIRT1 using shRNA also barely affected the proliferation and sphere formation of GB2 cells (Fig EV3F). These results suggest that SIRT2, but not SIRT1, plays an important role in the proliferation and survival of glioblastoma cells. Click here to expand this figure. Figure EV3. The SIRT2-specific inhibitor AGK2 induces growth arrest of glioblastoma cells (related to Fig 1) Salermide treatment decreases the proliferation of adherent GB2 cells. 5 × 104 cells were treated with 20 μM salermide or DMSO, and the number of viable cells was counted at the indicated time points. Bars indicate mean ± SD (n = 3). Dose–response curves for AGK2, salermide, or EX527 treatment on GB2 cells. 5 × 104 cells were treated with each reagent, respectively, at the indicated concentrations. After 72 h, the number of viable cells was counted. Bars indicate mean ± SD (n = 3). EX527 does not inhibit proliferation of GB2 cells. 5 × 104 cells were treated with AGK2 (20 μM), salermide (20 μM), or EX527 (1 or 20 μM) for 72 h. Bars indicate mean ± SD of triplicate technical repeats. EX527 does not inhibit sphere formation of GB2 cells. Cells were plated in 96-well plate (1.0 × 103 cells per well) and treated with AGK2 (20 μM), salermide (20 μM), or EX527 (1 or 20 μM). After 7 days of incubation, the number of spheres was counted. Bars indicate mean ± SD of 10 wells. EX527 does not increase the expression of PUMA or GADD45 mRNA. 1.5 × 105 cells were treated with AGK2 (20 μM), salermide (20 μM), or EX527 (1 or 20 μM), respectively, and subjected to qRT–PCR. Bars indicate mean ± SD (n = 3–4). GB2 cells were transfected with the indicated shRNAs. (Left panel) Immunoblotting analysis of the effect of shRNA on SIRT1 expression. (Middle panel) The number of viable cells was counted. (Right panel) The expression of PUMA was measured by qRT–PCR. Bars indicate mean ± SD (n = 3). Data information: Statistical significance was evaluated using unpaired two-tailed t-test. *P < 0.05; **P < 0.01; N.S., not significant. Source data are available online for this figure. Download figure Download PowerPoint Knockdown of SIRT2 suppresses the tumorigenicity of glioblastoma cells To examine the effect of SIRT2 knockdown on the tumorigenicity of glioblastoma cells, we transplanted GB2 cells in which SIRT2 expression had been knocked down into the frontal lobe of immunocompromised mice. All of the mice transplanted with control GB2 cells started to decline after 3 months and died within 4 months (Fig 2A). By contrast, all except for one of the mice transplanted with SIRT2-knockdown GB2 cells survived for more than 5 months after transplantation and only two mice showed obvious clinical symptoms of declining health (Fig 2A). We confirmed these results by a quantitative qRT–PCR measure of tumor burden using primers specific for human versus mouse β-actin (Fig 2B). Consistent with the results in GB2 cells, knockdown of SIRT2 also significantly suppressed the tumorigenicity of GB16 (mutated p53) cells (Fig 2A). On the other hand, SIRT2 knockdown had modest effect on GB13 (wild-type p53). Histological studies revealed that all mice transplanted with GB16 cells had developed tumors with diffuse infiltration into surrounding brain tissues, one of the hallmark features of glioblastoma (Fig 2C). By contrast, we found no obvious pathological lesions in the brains of the mice transplanted with SIRT2-knockdown GB16 cells. Figure 2. Knockdown of SIRT2 suppresses the tumorigenicity of GB2 cells Kaplan–Meier overall survival curves of mice transplanted with GB2 cells (left panel), GB13 cells (Middle panel) or GB16 cells (right panel) infected with the indicated lentivirus (1 × 104 cells, 7 mice per group). The y-axis indicates the percent survival. Mice were transplanted with the indicated number of GB2 cells infected with a control (empty) or shSIRT2-expressing (shS2 #1) lentivirus. Six weeks after transplantation, mice (3 or 4 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT–PCR. H&E staining of tumors that were developed in mice implanted with GB16 cells that had been infected with a control (empty) or shSIRT2-expressing (shS2#1) lentivirus. Scale bar, 1 mm. An image with higher magnification is shown on the right (scale bar, 50 μm). The effect of AK7 on the deacetylation of acetyl-tubulin (left panel) and the proliferation of GB2 cells (right panel). (Left panel) Immunoblotting analysis of the effect of AK7 (20 μM) on deacetylation of acetyl-tubulin/tubulin was performed on day 3 in right panel. Bars indicate mean ± SD (n = 3). Ten days after intracranial transplantation of GB2 cells (1.0 × 104 cells), AK7 was intraperitoneally administrated for 4 weeks (15 mg/kg, twice/week). After 8 weeks, mice (4 or 5 animals, see number of dots) were sacrificed and the expression levels of human β-actin mRNA were quantified by qRT–PCR. Data information: Statistical significance was evaluated using the log-rank test (for panel A) or unpaired two-tailed t-test. **P < 0.01. Source data are available online for this figure. Source Data for Figure 2 [embr201745587-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint We next investigated whether a small-molecule SIRT2 inhibitor could suppre" @default.
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• W2890593886 title "<scp>SIRT</scp> 2‐mediated inactivation of p73 is required for glioblastoma tumorigenicity" @default.
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