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• W2891660372 abstract "Article20 September 2018Open Access Transparent process Tuberous sclerosis complex is required for tumor maintenance in MYC-driven Burkitt's lymphoma Götz Hartleben Götz Hartleben European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Christine Müller Christine Müller European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Andreas Krämer Andreas Krämer Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Heiko Schimmel Heiko Schimmel Institute for Pathology, Jena University Hospital, Jena, Germany Search for more papers by this author Laura M Zidek Laura M Zidek Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Carsten Dornblut Carsten Dornblut Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author René Winkler René Winkler Center for Molecular Biomedicine, Friedrich Schiller University, Jena, Germany Search for more papers by this author Sabrina Eichwald Sabrina Eichwald Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Gertrud Kortman Gertrud Kortman European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Christian Kosan Christian Kosan orcid.org/0000-0002-8387-3653 Center for Molecular Biomedicine, Friedrich Schiller University, Jena, Germany Search for more papers by this author Joost Kluiver Joost Kluiver Department of Pathology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Iver Petersen Iver Petersen Institute for Pathology, Jena University Hospital, Jena, Germany Search for more papers by this author Anke van den Berg Anke van den Berg Department of Pathology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Zhao-Qi Wang Zhao-Qi Wang orcid.org/0000-0002-8336-3485 Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Cornelis F Calkhoven Corresponding Author Cornelis F Calkhoven [email protected] orcid.org/0000-0001-6318-7210 European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Götz Hartleben Götz Hartleben European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Christine Müller Christine Müller European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Andreas Krämer Andreas Krämer Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Heiko Schimmel Heiko Schimmel Institute for Pathology, Jena University Hospital, Jena, Germany Search for more papers by this author Laura M Zidek Laura M Zidek Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Carsten Dornblut Carsten Dornblut Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author René Winkler René Winkler Center for Molecular Biomedicine, Friedrich Schiller University, Jena, Germany Search for more papers by this author Sabrina Eichwald Sabrina Eichwald Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Gertrud Kortman Gertrud Kortman European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Christian Kosan Christian Kosan orcid.org/0000-0002-8387-3653 Center for Molecular Biomedicine, Friedrich Schiller University, Jena, Germany Search for more papers by this author Joost Kluiver Joost Kluiver Department of Pathology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Iver Petersen Iver Petersen Institute for Pathology, Jena University Hospital, Jena, Germany Search for more papers by this author Anke van den Berg Anke van den Berg Department of Pathology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Search for more papers by this author Zhao-Qi Wang Zhao-Qi Wang orcid.org/0000-0002-8336-3485 Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Cornelis F Calkhoven Corresponding Author Cornelis F Calkhoven [email protected] orcid.org/0000-0001-6318-7210 European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany Search for more papers by this author Author Information Götz Hartleben1,2, Christine Müller1,2, Andreas Krämer2, Heiko Schimmel3, Laura M Zidek2, Carsten Dornblut2, René Winkler4, Sabrina Eichwald2, Gertrud Kortman1, Christian Kosan4, Joost Kluiver5, Iver Petersen3, Anke van den Berg5, Zhao-Qi Wang2 and Cornelis F Calkhoven *,1,2 1European Research Institute for the Biology of Ageing, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands 2Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany 3Institute for Pathology, Jena University Hospital, Jena, Germany 4Center for Molecular Biomedicine, Friedrich Schiller University, Jena, Germany 5Department of Pathology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands *Corresponding author. Tel: +31 6 52 72 45 91; Fax: +31 50 361 73 10; E-mail: [email protected] The EMBO Journal (2018)37:e98589https://doi.org/10.15252/embj.201798589 PDFDownload PDF of article text and main figures. 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 The tuberous sclerosis complex (TSC) 1/2 is a negative regulator of the nutrient-sensing kinase mechanistic target of rapamycin complex (mTORC1), and its function is generally associated with tumor suppression. Nevertheless, biallelic loss of function of TSC1 or TSC2 is rarely found in malignant tumors. Here, we show that TSC1/2 is highly expressed in Burkitt's lymphoma cell lines and patient samples of human Burkitt's lymphoma, a prototypical MYC-driven cancer. Mechanistically, we show that MYC induces TSC1 expression by transcriptional activation of the TSC1 promoter and repression of miR-15a. TSC1 knockdown results in elevated mTORC1-dependent mitochondrial respiration enhanced ROS production and apoptosis. Moreover, TSC1 deficiency attenuates tumor growth in a xenograft mouse model. Our study reveals a novel role for TSC1 in securing homeostasis between MYC and mTORC1 that is required for cell survival and tumor maintenance in Burkitt's lymphoma. The study identifies TSC1/2 inhibition and/or mTORC1 hyperactivation as a novel therapeutic strategy for MYC-driven cancers. Synopsis Although generally associated with tumor suppression, the negative mTOR regulators TSC1/2 are appropriated by the MYC oncogene to prevent mTORC1 hyperactivation that may otherwise jeopardize survival of MYC-driven cancers. The Tuberous sclerosis complex (TSC) is highly expressed in MYC-driven Burkitt′s Lymphoma (BL) and is required for BL cell survival. MYC maintains high TSC1/2 expression in BL cells through transcriptional and post-transcriptional upregulation of TSC1. In combination with high MYC expression in BL, mTORC1 hyperactivation upon TSC loss-of-function leads to elevated mitochondrial respiration, toxic ROS production, and apoptosis. The tumor maintenance function of TSC in BL questions a general role of the TSC as a tumor suppressor. Introduction TSC1/2 is a critical upstream regulator of the mechanistic target of rapamycin complex (mTORC) 1 kinase. TSC1 (hamartin) stabilizes TSC2 (tuberin), which is the GTPase-activating protein (GAP) for Rheb (Ras homolog enriched in brain) in the regulation of mTORC1 (Mieulet & Lamb, 2010). Germline mutations in either TSC1 or TSC2 result in the development of benign tumors (hamartomas) due to hyperactive mTORC1 signaling, which, however, usually does not result in malignancy. Moreover, loss of function of either TSC1 or TSC2 is rarely found in malignant tumors (Mieulet & Lamb, 2010) with some known exceptions like somatic mutations of TSC1 in bladder cancer (Pymar et al, 2008) or TSC2 in hepatocellular carcinoma's (Huynh et al, 2015). Therefore, retaining functional TSC1/2-mTORC1 regulation may be beneficial for certain cancer cells (Mieulet & Lamb, 2010). An important role of TSC1/2 in metabolic homeostasis was revealed in hematopoietic stem cells (HSCs) where deletion of TSC1 results in elevated mTORC1 activity and increased ROS production with detrimental effects on HCS function and survival (Chen et al, 2008). A database survey revealed that TSC1-mRNA expression is the highest in Burkitt's lymphoma-derived cell lines compared to 36 different tumor-type cell lines in the Cancer Cell Line Encyclopedia (CCLE; http://www.broadinstitute.org/ccle) (Fig EV1A). Moreover, the COSMIC database (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/) lists no mutations for TSC1 or TSC2 in Burkitt's lymphoma and they are not under the recurrently mutated genes in Burkitt's lymphoma identified by genomic approaches in two studies (Richter et al, 2012; Schmitz et al, 2012). Burkitt's lymphoma is an aggressively growing malignancy characterized by a MYC translocation that induces very high expression levels of the proto-oncogenic transcription factor MYC (Molyneux et al, 2012). MYC promotes cell proliferation by multiple mechanisms including stimulation of cell cycle progression, ribosome biogenesis, tRNA synthesis, translation and metabolic adjustments to increase provision of metabolic intermediates (Dang, 2012). Click here to expand this figure. Figure EV1. TSC1 is highly expressed in Burkitt's lymphoma Box plots showing the relative TSC1 mRNA expression levels across different cancer cell line types with the horizontal line showing the median, whiskers showing upper and lower non-outlier limits, the box representing the first to the third quartiles, and open circles representing outliers. Data extracted from CCLE_Expression_Entrez_2012-10-18.res, with gene-centric robust multi-array analysis (RMA)-normalized mRNA expression data (the number of different cell lines is indicated in parentheses). TSC1 protein reduction precedes TSC2 reduction following repression of MYC (+Tet, 24 h) in P493-6 cells. Immunoblots showing expression levels of MYC, TSC1, TSC2, or α-tubulin in low (+Tet) versus high MYC (−Tet) P493-6 cells (in comparison with 72 h MYC repression shown in Fig 1B). Download figure Download PowerPoint In this study, we reveal that MYC stimulates the expression of the mTORC1-inhibitor TSC1 by a feed-forward mechanism combining TSC1 transcriptional activation and alleviation of microRNA miR-15a-mediated repression. Loss of TSC1 function in Burkitt's lymphoma cells results in enhanced mitochondrial respiration and accumulation of toxic ROS levels. Our study is the first to provide evidence that TSC1 has tumor maintenance function designating the TSC1/2-mTORC1 axis as a novel therapeutic target in MYC-driven Burkitt's lymphoma. Results MYC controls mTORC1 through upregulation of TSC1/2 in Burkitt's lymphoma To examine a potential MYC-TSC1 regulation in Burkitt's lymphoma (BL), we analyzed TSC1/2 expression in human BL cell lines, which express high levels of MYC, in comparison with low MYC expressing Hodgkin lymphoma (HL) cell lines. Immunoblotting revealed that high expression of TSC1/2 correlates with high MYC expression in BL cells and that low TSC1/2 expression correlates with low MYC in HL cells (Fig 1A). To investigate MYC-TSC1/2-mTORC1 regulation, we used the EBV immortalized human B-cell line P493-6 that carries a conditional, tetracycline-repressible MYC allele to study MYC-induced B-cell proliferation (Pajic et al, 2000). Also in this system, high MYC levels correlate with high TSC1/2 levels, and suppression of MYC (+Tet, 72 h) resulted in a reduction of TSC1 and TSC2 (Fig 1B). Quantitative real-time PCR (qRT–PCR) analysis revealed a strong reduction of TSC1 mRNA versus a minor reduction of TSC2 mRNA following 24-h repression of MYC (+Tet; Fig 1C). In addition, the decline in TSC1 protein occurred prior to the TSC2 reduction at the earlier 24-h time point (Fig EV1B). Since TSC1 stabilizes TSC2, these data suggest that low MYC levels primarily affect TSC1 expression followed by destabilization of TSC2. TSC1/2 is the major inhibitor of mTORC1 signaling and accordingly expression of high levels of MYC (−Tet) in P493-6 cells resulted in a strong reduction of phosphorylation of the mTORC1 substrate p70-S6-kinase1 (S6K) and its substrate ribosomal protein S6 measured over 24–72 h (Fig 1D). Knockdown of TSC1 in MYC expressing P493-6 (−Tet) resulted in lower levels of TSC2 and in stimulation of mTORC1 signaling, revealing integral MYC-TSC1/TSC2-mTORC1 regulation (Fig 1E). The phosphorylation of S6K and S6 in the low MYC (+Tet) cells is abrogated by rapamycin showing that the observed effects are mTORC1 linked (Fig 1F). Figure 1. MYC controls mTORC1 signaling through regulation of the TSC1 Immunoblot of expression levels of MYC, TSC1, TSC2, and β-actin loading control in high MYC Burkitt's lymphoma (BL) cells compared to low MYC Hodgkin lymphoma (HL) cells. Immunoblots showing expression levels of MYC, TSC1, TSC2, or β-actin loading control in P493-6 cells treated with tetracycline for 72 hours (+Tet) or in untreated cells (−Tet). Relative TSC1 and TSC2 mRNA expression levels determined by qRT–PCR for high MYC (−Tet) versus low MYC (+Tet) P493-6 cells treated for 24 h with tetracycline (mean ± SD, n = 3 technical replicates). *P < 0.05; **P < 0.01; statistical relevance was determined by unpaired t-test (two-tailed). qRT–PCR analysis of TSC1 mRNA levels upon MYC suppression for 24 h–72 h (+Tet). Immunoblots for 24 h and 48 h (+Tet) show S6K and phosphorylation (P-) of S6K as downstream mTORC1 target, and β-actin loading control. For 72 h (+Tet), the immunoblots show expression of MYC and phosphorylation (P-) of downstream mTORC1 targets S6K and S6, and α-tubulin as loading control. Upper immunoblot shows the reduction in TSC1 levels upon expression of two different TSC1-specific shRNAs compared to scrambled control shRNA in P493-6 cells. Other blots show the expression levels of TSC2, S6K/P-S6K, S6/P-S6, and α-tubulin for loading control. Immunoblots of indicated proteins in P493-6 cells with high MYC (−Tet, 72 h) or low MYC (+Tet, 72 h) levels either treated with rapamycin or solvent. Download figure Download PowerPoint Next, we analyzed the expression of MYC and TSC1 by immunohistochemistry of human BL tissue samples versus control reactive lymph node tissue samples. We found significantly higher expression of MYC and TSC1 protein levels in the BL samples compared to the B lymphocytes that reside in the germinal centers of control lymph nodes (Figs 2A and EV2A and E). In addition, in a second cohort of BL patient samples, we found significantly higher expression of TSC1 and TSC2 proteins by immunoblotting in BL compared to control tonsils and reactive lymph nodes (LN) (Figs 2B and EV2B). Thus, our results show that high TSC1/2 expression correlates with high MYC expression in BL and BL cell lines. Finally, S6K-phosphorylation levels as determined by immunoblotting were lower for BL patient samples compared to reactive lymph node controls (Fig EV2C). S6-phosphorylation was virtually absent in seven BL tissue samples and present in one BL sample. In the reactive control lymph nodes, S6-phosphorylation staining was observed in a mosaic fashion and with different intensities (Fig EV2D and F). Figure 2. Levels of TSC1 and MYC correlate in Burkitt's lymphoma Elevated TSC1 and MYC expression in BL (cohort 1). Example of immune staining of TSC1, MYC, the B-cell marker CD20, and DAPI nuclear DNA-staining in germinal centers of control lymph nodes (upper rows) and samples from BL patients (lower rows). Boxplots at the right show quantification of TSC1 or MYC staining from control germinal centers and BL samples (see materials and methods; the horizontal line shows the median, whiskers show maximum and minimum data points, and the box represents the first to the third quartiles, n = 56 fields for tumor samples and n = 21 fields for control germinal centers; scale bar = 100 μm). Quantification of immunoblot analysis of TSC expression in BL (cohort 2) (n = 13) compared to healthy tonsils (n = 3) or reactive lymph nodes (n = 3) (mean ± SD) shown in Fig EV2B. Data information: In all graphs *P < 0.05; **P < 0.01; ***P < 0.001, statistical relevance was determined by unpaired t-test (two-tailed). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Reduced activity of the mTORC1 pathway in BL patient samples H&E-stained pictures of the samples in Figs 2A and EV2D. Two representative sections of the indicated sample are presented in 100× (left) and 200× (right) magnification, scale bar = 100 μm. Immunoblot of Fig 2B. Frozen tissue slices of individual Burkitt's lymphoma (BL), reactive lymph nodes (LN), or tonsils were lysed and subjected to immunoblotting with the indicated antibodies. Quantification shown in Fig 2B was done with ImageJ. Immunoblot of the same samples as in (B) with the indicated antibodies. Graph at the right shows the quantification of the P-S685K/S6K ratio performed with ImageJ (mean ± SD, n = 3 for reactive LN, n = 10 for lymphomas). ***P < 0.001, statistical relevance was determined by unpaired t-test (two-tailed). Example of immune staining of P-S6, the B-cell marker CD20, and DAPI nuclear DNA-staining in control lymph nodes (upper row) and Burkitt's lymphoma (lower rows). Of eight investigated lymphoma samples, we found seven with virtual absence of P-S6 staining and one with positive P-S6 staining. The samples belong to the same cohort used in Figs 2A and EV2A, scale bar = 100 μm. Wild-type (wt) MEFs and TSC1-deficient MEFs immunostained with anti-TSC1 antibody to confirm its specificity. Scale bar, 50 μm. wt MEFs untreated and wt MEFs treated with 20 nM rapamycin for 12 h were immunostained with anti-phosphorylated-S6 antibody to confirm its specificity. Scale bar, 50 μm. Download figure Download PowerPoint Altogether, our data show that TSC1/2 expression is remarkably high in MYC BL systems and suggesting that during oncogenesis MYC maintains control of mTORC1 signaling through stimulation of TSC function. Loss of TSC1 function is lethal for MYC-driven cancer cells Given the anticipated role of TSC1 as a tumor suppressor, these rather unexpected findings led us to examine whether TSC1 upregulation is required for the oncogenic potential of MYC in the cellular BL model. Strikingly, TSC1 knockdown in high (−Tet) MYC P493-6 cells resulted in a strong decrease in viable cell numbers (Fig 3A, left graph). AnnexinV/7AAD staining revealed that apoptosis was increased in TSC1 knockdown cells (Fig 3A, right graph), suggesting that the upregulation of TSC1 by MYC is required for cell survival. Notably, the decreased P493-6 cell viability in response to TSC1 knockdown could be rescued by treatment with the mTORC1 inhibitor rapamycin, showing that enhanced mTORC1 activity is responsible for the increased apoptosis (Fig 3B). To further analyze a potential synthetic lethal interaction between MYC deregulation and mTORC1 hyperactivation, we made use of an U2OS cell line expressing a MYC-ER fusion protein (Liu et al, 2012). In cells with physiological MYC activity (−OHT), the activation of mTORC1 through knockdown of TSC1 had little effect on cell viability. However, the combined activation of MYC (+OHT) and mTORC1 (TSC1 knockdown) synergistically increased apoptosis, which could be prevented with rapamycin treatment (Figs 3C and EV3A). This shows that cells with deregulated MYC require restriction of mTORC1 signaling for survival and that TSC inhibition is synthetic lethal with MYC overexpression. Figure 3. TSC1 is crucial for survival of Burkitt's lymphoma (BL) cells Left graph shows the multiplication rate of P493-6 (−Tet) cells expressing either a scrambled control shRNA or a TSC1-specific shRNA determined by viable cell counting 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± SD, n = 3 biological replicates). Right graph shows percentage of apoptotic P493-6 (−Tet) cells expressing scrambled control shRNA or a TSC1-specific shRNA determined by FACS analysis of AnnexinV/7AAD-stained cells (mean ± SD, n = 3 biological replicates). Rapamycin treatment recovers survival of TSC1 knockdown in P493-6 cells. Relative viable cell number counts of P493-6 (−Tet) cells expressing scrambled control shRNA or TSC1-specific shRNA 14 days after seeding equal number of viable cells (Trypan blue exclusion), in the presence of 30 pM rapamycin where indicated (mean ± SD, n = 3 biological replicates). TSC1 knockdown is synthetic lethal with MYC deregulation. U2OS-MYC-ER cells expressing either scrambled control shRNA or TSC1-specific shRNA were treated with hydroxytamoxifen (4-OHT) to induce MYC and rapamycin (100 nM) where indicated. Percentage of apoptotic cells was determined with Annexin/7AAD staining 4 days after MYC induction (mean ± SD, n = 3 biological replicates). Survival rate of different BL cell lines upon TSC1 knockdown. Graphs show numbers of viable cells expressing either a scrambled control shRNA or a TSC1-specific shRNA 3 days after seeding of equal number of viable cells (determined by Trypan blue exclusion; mean ± SD, n = 3 biological replicates). Immunoblots of control- or TSC1-shRNA expressing BL2 or DG75 cells treated with different concentrations of rapamycin to either completely inhibit mTORC1 activity (10 nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± SD, n = 3 biological replicates); (BL2 cells were selected for stable knockdown with puromycin, DG75 cells without selection). Ramos cells expressing either a TSC1-specific or a control shRNA were inoculated into NOD/SCID mice, and tumor volume was measured regularly. The immunoblot shows the level of knockdown of TSC1 (sh-TSC1b) compared to control (sh-contr), and levels of TSC2 and phosphorylated mTORC1 target proteins before inoculation. Tumor growth curves show mean ± SEM (n = 8/group). Pictures of xenotransplanted human Ramos lymphoma tumors 35 days after inoculation. The top panels show tumors derived from Ramos cells stably expressing control shRNA (sh-contr); the lower panels show tumors derived from Ramos cells stably expressing TSC1-specific shRNA (sh-TSC1b). Data information: In all graphs *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001, statistical relevance was determined by unpaired t-test (two-tailed). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. TSC1 is required for survival of BL cells A. Immunoblots of the experiment in Fig 3C. U2OS-MYC-ER cells expressing scrambled control shRNA or TSC1-specific shRNA, treated with hydroxytamoxifen (4-OHT) or rapamycin where indicated. Immunoblots showing expression of TSC1, S6K/P-S6K, or α-tubulin. B–E. Immunoblots of the experiments in Fig 3D to confirm TSC1 knockdown. BL cells expressing scrambled control shRNA or TSC1-specific shRNA and subjected to immunoblotting with the indicated antibodies. The corresponding immunoblots for Raji and DG75 are shown in Fig EV4D and E, respectively. F. At the top, immunoblots of indicated proteins in Hodgkin lymphoma (HL) cell lines KMH2 and L540 expressing either a TSC1-specific shRNA or scrambled control shRNA. The bar graphs below show that TSC1 knockdown does not affect cell viability in KMH2 or L540 cells (mean ± SD, n = 3 biological replicates). See Fig 1A for MYC and TSC1 expression levels. G. Immunoblots show the reduction of TSC2 levels upon expression of a TSC2-specific shRNA compared to scrambled control shRNA in the indicated BL cell lines and in addition the expression levels of S6K/PS6K and β-actin as loading control. The bar graphs at the right show the relative cell viability of the same BL cell lines expressing either TSC2-specific shRNA or scrambled control shRNA 2 days after seeding equal amounts of viable cells as determined by a cell viability assay (mean ± SD, biological replicates n = 3 for Ramos, n = 5 for BL2 and n = 6 for CA46). H. Control- or TSC1-shRNA expressing Raji BL cells treated with different concentrations of rapamycin to either completely inhibit mTORC1 activity (10 nM) or titrate the activity to control levels (30 pM), and survival rate of these cells over 7 days (mean ± SD, n = 3 biological replicates). Data information: In all graphs **P < 0.01; ***P < 0.001, statistical relevance was determined by unpaired t-test (two-tailed). Download figure Download PowerPoint In order to have a better representation of the human disease, we studied the effect of TSC1 knockdown on viability for BL cell lines. TSC1 knockdown reduced cell viability of all eight tested BL cell lines (Fig 3D; see Figs EV3B–E and EV4D–E for TSC1 knockdown). TSC1 knockdown does not reduce cell viability in the low MYC and low TSC1 Hodgkin lymphoma (HL) cell lines KMH2 and L540 (Fig EV3F). The data suggest that high expression of TSC1 is a specific requirement for BL cells to maintain cell survival. To investigate whether loss of TSC2 as the other part of the TSC1/2 complex similarly affects cell survival, we knocked down TSC2 in three selected BL cell lines. In all three cell lines, TSC2 knockdown resulted in increased S6K-phosphorylation and reduced of cell viability (Fig EV3G). Our findings may seem to be at odds with studies showing that mTORC1 signaling is required for survival of lymphomas in the Eμ-Myc mouse model (Wall et al, 2013) or in TCF3-activated tonic BCR signaling that activates mTORC1 (Schmitz et al, 2012). Therefore, we hypothesized that maintaining control of mTORC1 to prevent hypo- as well as hyperactivity is important for Burkitt's lymphoma survival. To test this hypothesis, we performed a rapamycin titration experiment using the BL cell lines BL2, DG75, and Raji. As expected, strong inhibition of mTORC1 with a high dose of rapamycin (10 nM) severely reduced the cell viability in both control and TSC1 knockdown cells. In contrast, the decreased viability of the TSC1 knockdown cells was recovered by treatment with low dose of rapamycin (30 pM), which titrated the mTORC1 activity to comparable levels of that in control cells (Figs 3E and EV3H). Thus, a controlled level of mTORC1 activity that is in equilibrium with MYC expression levels is critical for the survival of BL cells and probably other MYC-driven cancer cells. Click here to expand this figure. Figure EV4. TSC1 knockdown increases mitochondrial function Immunoblots of control- or TSC1-shRNA expressing BL cells as indicated showing the effect of the TSC1 knockdown on phosphorylation of AKT (P-Ser473). Antibodies specific for AKT and β-actin served as controls. The incubation of the same immunoblots with an antibody specific for P-Thr308 AKT did not produce any detectable signal. TSC1 knockdown increases ratio of oxygen consumption to lactate production (as measured by acidification) in a rapamycin-dependent manner. Ratio of oxygen consumption to lactate production rates determined in the experiment of Figure 4A in high MYC P493-6 (−Tet) cells expressing scrambled control shRNA or TSC1-specific shRNA, treated with 20 nM rapamycin for 12 h where indicated (mean ± SD, n = 6 biological replicates). Relative mRNA expression determined by qRT–PCR of ATP5G1 (left graph) or cytochrome C (CYCS) (right graph) in Raji cells expressing scrambled control shRNA or TSC1-specific shRNA (mean ± SD, n = 3). From left to right in Raji cells expressing scrambled control shRNA or TSC1-specific shRNA: immunoblots showing expression of TSC1, S6K/P-S6K, or α-tubulin; rate of oxygen consumption, basal and in response to 10 μM DNP or 10 μM oligomycin where indicated; ratio of oxygen consumption to lactate production rates (mean ± SD, n = 8). From left to right in DG75 cells expressing scrambled control shRNA or TSC1-specific shRNA: immunoblots" @default.
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• W2891660372 date "2018-09-20" @default.
• W2891660372 modified "2023-09-26" @default.
• W2891660372 title "Tuberous sclerosis complex is required for tumor maintenance in MYC‐driven Burkitt's lymphoma" @default.
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