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• W2590751720 abstract "Article20 February 2017free access Source DataTransparent process MYC-driven inhibition of the glutamate-cysteine ligase promotes glutathione depletion in liver cancer Brittany Anderton Brittany Anderton Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Roman Camarda Roman Camarda Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Sanjeev Balakrishnan Sanjeev Balakrishnan Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Asha Balakrishnan Asha Balakrishnan Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, TWINCORE, Center for Experimental and Clinical Infection Research, Hannover, Germany Search for more papers by this author Rebecca A Kohnz Rebecca A Kohnz Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA Search for more papers by this author Lionel Lim Lionel Lim Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Kimberley J Evason Kimberley J Evason Department of Pathology and Huntsman Cancer Institute, University of Utah, Salt Lake, UT, USA Search for more papers by this author Olga Momcilovic Olga Momcilovic Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Klaus Kruttwig Klaus Kruttwig Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Qiang Huang Qiang Huang Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Guowang Xu Guowang Xu Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Daniel K Nomura Daniel K Nomura Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA Search for more papers by this author Andrei Goga Corresponding Author Andrei Goga [email protected] orcid.org/0000-0001-9127-0986 Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Brittany Anderton Brittany Anderton Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Roman Camarda Roman Camarda Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Sanjeev Balakrishnan Sanjeev Balakrishnan Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Asha Balakrishnan Asha Balakrishnan Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, TWINCORE, Center for Experimental and Clinical Infection Research, Hannover, Germany Search for more papers by this author Rebecca A Kohnz Rebecca A Kohnz Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA Search for more papers by this author Lionel Lim Lionel Lim Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Kimberley J Evason Kimberley J Evason Department of Pathology and Huntsman Cancer Institute, University of Utah, Salt Lake, UT, USA Search for more papers by this author Olga Momcilovic Olga Momcilovic Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Klaus Kruttwig Klaus Kruttwig Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Qiang Huang Qiang Huang Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Guowang Xu Guowang Xu Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China Search for more papers by this author Daniel K Nomura Daniel K Nomura Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA Search for more papers by this author Andrei Goga Corresponding Author Andrei Goga [email protected] orcid.org/0000-0001-9127-0986 Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA Department of Medicine, University of California, San Francisco, San Francisco, CA, USA Search for more papers by this author Author Information Brittany Anderton1,2, Roman Camarda1,2, Sanjeev Balakrishnan1,2, Asha Balakrishnan1,2,3, Rebecca A Kohnz4, Lionel Lim1,2, Kimberley J Evason5, Olga Momcilovic1,2, Klaus Kruttwig1,2, Qiang Huang6, Guowang Xu6, Daniel K Nomura4 and Andrei Goga *,1,2 1Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA, USA 2Department of Medicine, University of California, San Francisco, San Francisco, CA, USA 3Department of Gastroenterology, Hepatology, and Endocrinology, Hannover Medical School, TWINCORE, Center for Experimental and Clinical Infection Research, Hannover, Germany 4Department of Nutritional Sciences and Toxicology, University of California, Berkeley, CA, USA 5Department of Pathology and Huntsman Cancer Institute, University of Utah, Salt Lake, UT, USA 6Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian, China *Corresponding author. Tel: +1 415 476 4191; E-mail: [email protected] EMBO Reports (2017)18:569-585https://doi.org/10.15252/embr.201643068 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 How MYC reprograms metabolism in primary tumors remains poorly understood. Using integrated gene expression and metabolite profiling, we identify six pathways that are coordinately deregulated in primary MYC-driven liver tumors: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption. We then focus our attention on glutathione (GSH) and glutathione disulfide (GSSG), as they are markedly decreased in MYC-driven tumors. We find that fewer glutamine-derived carbons are incorporated into GSH in tumor tissue relative to non-tumor tissue. Expression of GCLC, the rate-limiting enzyme of GSH synthesis, is attenuated by the MYC-induced microRNA miR-18a. Inhibition of miR-18a in vivo leads to increased GCLC protein expression and GSH abundance in tumor tissue. Finally, MYC-driven liver tumors exhibit increased sensitivity to acute oxidative stress. In summary, MYC-dependent attenuation of GCLC by miR-18a contributes to GSH depletion in vivo, and low GSH corresponds with increased sensitivity to oxidative stress in tumors. Our results identify new metabolic pathways deregulated in primary MYC tumors and implicate a role for MYC in regulating a major antioxidant pathway downstream of glutamine. Synopsis This study shows that MYC inhibits GCLC, the rate-limiting enzyme of glutathione synthesis, via the microRNA miR-18a in primary tumors. Therefore, MYC-driven liver tumors with low total tissue levels of glutathione exhibit increased sensitivity to a potent oxidant. Six metabolic pathways deregulated by MYC in primary liver tumors are identified using integrated biochemical and gene expression analyses. Total glutathione (GSH) levels are depleted in MYC-driven liver tumors, which is associated with reduced glutathione synthesis. GCLC, the rate-limiting enzyme of GSH synthesis, is attenuated by the MYC-induced miR-18a. Exogenous treatment of primary MYC liver tumors with the oxidant diquat specifically induces tumor cell death and diminishes MYC expression in surviving tumor cells. Introduction Metabolic reprogramming is a hallmark of MYC-driven tumor growth and maintenance 1. Most notably, MYC orchestrates tumor cell dependence on glucose and glutamine for biomass accumulation 1234. MYC regulates many points in glycolysis through transcriptional regulation of glycolytic genes. For example, MYC regulates the GLUT1 transporter 5 and glycolytic enzymes such as LDHA 6 and PKM2 7. Expression of PKM2 in cancer has been suggested to enhance proliferative metabolism by causing a “backflow” of metabolites into anabolic pathways such as the serine biosynthesis pathway 8. MYC also regulates glutaminolysis at multiple nodes. Many MYC-overexpressing cell lines are addicted to glutamine 910 and primary MYC-driven murine lung and liver tumors display increased glutamine metabolism 11. Expression of the glutamine transporter Slc1a5 is regulated by MYC and is elevated in MYC-driven liver tumors 1112. MYC also activates expression of glutamine synthetase through promoter demethylation in some tumors 13. Glutaminase (GLS), the enzyme that catalyzes the conversion of glutamine to glutamate, is upregulated in lymphoma cells due to MYC-dependent inhibition of miR-23a/b expression 14. GLS inhibition in cell lines or xenograft tumors causes MYC-dependent cell death or reduced proliferation 111215. Elevated glutamine uptake and metabolism can contribute to TCA cycle flux and glutathione synthesis in MYC-expressing cells 121415. However, whether MYC regulates specific pathways downstream of glutamine conversion to glutamate in tumor cells remains unknown. Most work to uncover MYC's role in metabolism to date has been conducted using cultured tumor cells that are removed from the native tumor environment 34. However, cell culture conditions may not accurately reflect nutrient availability and uptake in the host tissue. In this study, we sought to identify novel metabolic pathways that are altered in MYC-driven primary tumors in vivo. MYC drives human hepatocarcinogenesis 16, is frequently amplified or overexpressed in human hepatocellular carcinoma (HCC), and is associated with poorly differentiated tumors and poor liver cancer prognosis 1718192021. We thus sought to characterize altered metabolism in a mouse model of MYC-driven liver cancer. We performed mRNA gene expression and mass spectrometry-based biochemical profiling of tumor samples from the LAP-tTA × TetO-MYC (LT2-MYC) bi-transgenic mouse, a conditional model that drives MYC expression and tumorigenesis specifically in hepatocytes 2223 (Fig 1A). We recently showed that this model has gene expression changes consistent with aggressive, poorly differentiated human liver cancers 24. Use of this model allows us to observe MYC-mediated metabolic reprogramming in the native tumor environment 23. Additionally, we can take advantage of its conditional nature to identify changes that are a direct effect of MYC signaling in vivo (Fig 1A). Our work identifies a novel role for MYC in regulating the synthesis of glutathione, a major cellular antioxidant, via miR-18a in primary tumors. This finding has implications for the use of oxidative stress-inducing drugs for therapy of MYC liver tumors. Figure 1. Integrated metabolic analysis of MYC-driven liver tumors Summary of LT2-MYC conditional transgenic mouse model of MYC-induced hepatocarcinogenesis. Prolonged MYC overexpression induces tumor nodules that are morphologically and histologically distinct from non-tumor tissue. MYC protein expression can be turned off in established tumors and correlates with alpha-fetoprotein (AFP) expression, a marker of aggressive liver cancer (see REG 7 day Western blot). In images, white arrows indicate non-tumor liver tissue and yellow arrows indicate liver tumor tissue. Scale bars in hematoxylin and eosin-stained (H&E) sections represent 20 μm. Transcriptional and biochemical profiling analyses identify six pathways that are significantly altered in LT2-MYC tumors versus control livers (n = 3 LT2 control and n = 4 LT2-MYC for transcriptional profiling, n = 7 in each group for biochemical profiling, Fisher's exact test, P < 0.05). Glutathione pathway (KEGG #ko00480) metabolite abundances segregate LT2-MYC tumors from control livers by unsupervised hierarchical clustering (n = 7 in each group, LT2 control liver samples in green, LT2-MYC tumor samples in gray). Source data are available online for this figure. Source Data for Figure 1 [embr201643068-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Results Integrated metabolic analysis of MYC-driven liver tumors To identify novel metabolic pathways that are altered in primary liver tumors with high MYC expression (Fig 1A), we performed mRNA expression and mass spectrometry-based metabolite profiling of LT2-MYC tumor samples and naïve LT2 liver controls (Fig 1B and Dataset EV1). Of 333 detected metabolites with KEGG PATHWAY database identifiers 25, 188 were significantly altered in LT2-MYC tumors versus control liver tissue (FDR < 0.05). Likewise, 3,706 genes with KEGG identifiers exhibited significant deregulation in LT2-MYC tumors versus controls (FDR < 0.05). We performed pathway enrichment analysis, referencing all metabolic pathways defined by KEGG, of the significantly altered transcripts and metabolites. We identified six KEGG pathways that were significantly altered, both transcriptionally and biochemically, in LT2-MYC tumors compared to control liver tissues: glutathione metabolism; glycine, serine, and threonine metabolism; aminoacyl-tRNA biosynthesis; cysteine and methionine metabolism; ABC transporters; and mineral absorption (Fig 1B and Appendix Figs S1–S6). Of the six KEGG pathways that were coordinately deregulated, glutathione metabolism exhibited the most significantly altered gene expression and the second highest rate of metabolite abundance change (Appendix Table S1). Glutathione (GSH) is an important cellular antioxidant synthesized from glutamine carbons 26. In accordance with our integrated analyses, altered glutathione pathway metabolites and transcripts readily segregate LT2-MYC tumors from LT2 control livers by unsupervised hierarchical clustering (Figs 1C and EV1A, respectively). Glutathione (GSH) and glutathione disulfide (GSSG) were among the most dramatically depleted metabolites profiled in the MYC-driven tumors; we independently confirmed the depletion of total glutathione (GSH + GSSG) in the same MYC liver tumor samples using an enzymatic assay (Fig 2A). Interestingly, the depletion of GSH and GSSG was observed in murine liver tumors driven by MYC but not in those driven by RAS (LT2-RAS model described in 24) (Fig EV1B and C). This indicates that GSH depletion is not due to liver tumorigenesis in general, but instead is MYC oncogene-specific. Click here to expand this figure. Figure EV1. Glutathione metabolism is altered in MYC-driven liver tumors A. Glutathione pathway transcript expression segregates LT2-MYC tumors from control livers by unsupervised hierarchical clustering (n = 3 control livers in green, n = 4 tumors in gray). B, C. Relative metabolite abundance of GSH (B) or GSSG (C) in tissue samples from murine liver tumors driven by MYC or RAS, as compared to normal liver controls (n = 7 control liver, n = 7 MYC tumor, n = 7 RAS tumor, data represented as box plots with horizontal bar representing the median, box ranges representing the first (bottom) and third (top) quartiles, and vertical bars representing the standard error, unpaired two-tailed t-test, Bonferroni adjusted P-value for multiple comparisons is 0.02). Download figure Download PowerPoint Figure 2. Characterization of aberrant glutathione metabolism in MYC-driven liver tumors Total glutathione (GSH + GSSG) measured by enzymatic assay in LT2-MYC tumors versus control livers (n = 5 LT2 control samples, n = 6 LT2-MYC tumor samples, data represented as mean ± SEM, unpaired two-tailed t-test, P = 0.006). Multiple metabolites and enzymes in the glutathione metabolism pathway are significantly altered in LT2-MYC tumors versus control livers (unpaired two-tailed t-test, P < 0.1). Red = significantly elevated at P < 0.1, blue = significantly depleted at P < 0.05, and red and blue asterisks indicate that individual gamma-glutamyl amino acids are significantly increased or decreased at P < 0.05. Increased protein expression of the GLS1 isoform of glutaminase was previously reported for LT2-MYC tumors 11. Gamma-glutamylcysteine abundance in MYC-driven tumors as compared to adjacent non-tumor tissue (n = 6 each group, data represented as normalized mean ± SEM, paired one-tailed t-test, P = 0.04). Western blot analysis of key enzymes involved in the glutathione metabolism pathway in LT2-MYC tumors versus non-tumor LT2 controls (n = 2–3 each as indicated in images, unpaired two-tailed t-test on normalized expression, GSS P = 0.7, GLRX5 #P = 0.09, GGT1 *P = 0.05, GSR ***P = 0.0004, G6PDH **P = 0.001, GCLC ***P = 0.0004). For GCLC, LT2-MYC tumors regressed for 7 days by feeding doxycycline chow are also shown. Relative incorporation of [U-13C]-glutamine into gamma-glutamylcysteine and GSH in MYC-driven tumors compared to adjacent non-tumor liver tissue (n = 6 each group, data represented as normalized mean ± SEM, unpaired two-tailed t-test, gamma-glutamylcysteine P = 0.03, GSH P = 0.004). Source data are available online for this figure. Source Data for Figure 2 [embr201643068-sup-0005-SDataFig2.pdf] Download figure Download PowerPoint Characterization of aberrant glutathione metabolism in MYC-driven liver tumors We next sought to identify likely reasons for GSH depletion in MYC-driven liver tumors. Decreased GSH synthesis, increased gamma-glutamyl cycling, and increased protein S-glutathionylation may all contribute to low tissue GSH 26. Our metabolic profiling found that abundance of glutamine, GSH, and GSSG decreased while abundance of glutamate, cysteine, and glycine increased in MYC tumors relative to non-tumors (Fig 2B and Appendix Fig S1B). Decreased glutamine concomitant with elevated glutamate is consistent with elevated GLS expression and activity and has been previously described in LT2-MYC tumors 11. We performed a separate metabolic analysis and found that gamma-glutamylcysteine, the direct precursor of GSH, is depleted in MYC-driven tumors relative to non-tumor tissue (Fig 2B and C). Taken together, these data indicate a bottleneck in GSH production, particularly at the step catalyzed by glutamate-cysteine ligase, catalytic subunit (GCLC) (Fig 2B). Our metabolic profiling did not strongly suggest that gamma-glutamyl cycling was elevated in MYC tumors relative to non-tumor controls. Although 5-oxoproline was significantly elevated and multiple individual gamma-glutamyl amino acids were significantly altered in MYC tumors relative to non-tumors, the gamma-glutamyl amino acids were not uniformly altered—some were elevated and some were depleted (Figs 2B and EV2A). Further, we did not find evidence for elevated S-glutathionylation in the tumors. Our metabolic profiling indicated that both cysteine–glutathione disulfide and S-methylglutathione were dramatically depleted in MYC-driven liver tumors relative to non-tumor controls (Fig EV2B). Taken together, our biochemical profiling data strongly suggest that impairment of GSH production, rather than elevated gamma-glutamyl cycling or S-glutathionylation, contributes to GSH loss in the MYC-driven tumors. Click here to expand this figure. Figure EV2. Characterization of gamma-glutamyl amino acid and S-glutathionylation abundance in MYC-driven liver tumors Metabolite profiling of gamma-glutamyl amino acids in LT2-MYC tumors versus control livers (n = 7 control liver, n = 7 MYC tumor, data represented as normalized mean ± SEM, unpaired two-tailed t-test, *P < 0.05, **P < 0.01, ****P < 0.00001). Metabolite profiling of cysteine–glutathione disulfide and S-methylglutathione in LT2-MYC tumors versus control livers (n = 7 control liver, n = 7 MYC tumor, data represented as normalized mean ± SEM, unpaired two-tailed t-test, cysteine–glutathione disulfide P = 1.97305E-07, S-methylglutathione P = 1.73948E-09). Download figure Download PowerPoint We next sought to characterize the expression of enzymes that regulate GSH metabolism (Fig 2B). We performed Western blot analysis to determine the protein expression of several key GSH pathway enzymes, including GCLC; glutathione synthetase (GSS); gamma-glutamyltransferase 1 (GGT1); glutaredoxin 5 (GLRX5); glutathione reductase (GSR); and glucose-6-phosphate dehydrogenase (G6PDH) (Fig 2D). Our Western blot analysis indicated that protein expression of GLRX5, GGT1, GSR, and G6PDH increased (P ≤ 0.05 for GGT1, GSR, G6PDH; 0.05 < P < 0.10 for GLRX5), expression of GCLC markedly decreased (P < 0.001), and expression of GSS did not change in MYC-driven liver tumors compared to naïve liver tissue (Fig 2B and D). Although significant, the increase in GGT1 expression in tumors was very small. On the other hand, substantial downregulation of GCLC, the rate-limiting enzyme of GSH synthesis, is consistent with our hypothesis that GSH synthesis is impaired in tumors. Taken together, our metabolomic and Western blot data strongly suggest that decreased GSH synthesis contributes to depletion of free GSH in LT2-MYC tumors. However, we could not entirely rule out contributions from elevated gamma-glutamyl cycling and glutaredoxin activity. Isotopic tracing of glutamine in MYC-driven liver tumors MYC activates expression of the glutamine transporter Slc1a5, which increases cellular uptake of glutamine 1112. GSH is synthesized downstream of the conversion of glutamine to glutamate by GLS, which is elevated in a MYC-dependent manner 27 (Fig 2B). Previous studies suggest that elevated glutamine uptake contributes to GSH synthesis in MYC-overexpressing cells 121415. Because we saw depleted GSH concomitant with decreased GCLC expression and increased abundance of several GSH precursors in LT2-MYC tumors, we sought to trace the flow of glutamine-derived carbons in MYC-driven liver tumors to confirm whether GSH synthesis is impaired. We used mice from a somatic transgenic model of MYC-driven liver tumorigenesis 28, which also have elevated MYC and depleted GCLC protein expression (Fig EV3A). Tumor-bearing mice were injected with fully labeled [U-13C]-glutamine and mass spectrometry-based isotopic tracing of liver tumors was performed and compared to adjacent non-tumor liver tissue (Fig EV3B and C). We observed decreased incorporation of [U-13C]-glutamine carbons into GSH and γ-glutamyl-cysteine in tumors relative to adjacent non-tumor liver tissue (Figs 2E and EV3C). This is consistent with the diminished steady state abundances of GCLC protein (Figs 2D and EV3A) and GSH and GSSG metabolites we observed (Fig 2A). These results, together with the unchanged expression of GSS in tumors (Fig 2B and D), indicate that GSH synthesis via GCLC is impaired in MYC-driven liver tumors. Click here to expand this figure. Figure EV3. Isotopic tracing of glutamine in MYC-driven liver tumors Western blot analysis of MYC and GCLC protein expression in MYC-driven murine liver tumors established by hydrodynamic transfection 28 (n = 4 adjacent non-tumor (ANT), n = 4 MYC tumor). Schematic summary of U-[13C]-glutamine tracing experiment in MYC-driven liver tumor-bearing mice. Relative incorporation of [13C]-glutamine into metabolites in MYC-driven tumors compared to adjacent non-tumor liver tissue (n = 6 adjacent non-tumor (NT), n = 6 MYC tumor (T), all data represented as normalized mean ± SEM, unpaired two-tailed t-test, all data significant at P < 0.05). Source data are available online for this figure. Download figure Download PowerPoint GCLC expression is attenuated by miRNA-18a in MYC-driven liver tumors Because MYC regulates numerous genes involved in tumor metabolism, we reasoned that MYC might also regulate GCLC expression to control GSH synthesis. In support of this hypothesis, we found that GCLC protein and mRNA exhibit an inverse correlation with MYC signaling in vivo. Both GCLC protein (Fig 2D) and transcript (Fig EV4A) are low in tumor tissues relative to non-tumor tissue, and return to baseline when MYC is turned off in tumors by ad libitum doxy chow feeding (Fig 1A). Additionally, we find that total GSH levels increase in some tumors upon 72 h tumor regression, relative to tumor tissue (Fig EV4B). We further observed MYC-dependent changes in GCLC protein expression in a murine liver tumor cell line derived from the LT2-MYC model 29. When cells are grown in the presence of 8 ng/ml doxycycline, MYC expression is rapidly inhibited (Fig 3A). Using this conditional system, we found that GCLC protein increases when MYC is conditionally turned off over several days (Fig 3A). Click here to expand this figure. Figure EV4. GCLC expression and activity are altered in MYC-driven liver tumors Quantitative PCR analysis of Gclc mRNA expression in LT2-MYC tumors, control livers, and liver tumors regressed for 72 h (n = 4 each group, data represented as univariate scatter plot with median, unpaired two-tailed t-test, LT2 ctrl versus LT2-MYC P = 0.0003, LT2-MYC versus 72 h regression P = 0.0009). Total glutathione (GSH + GSSG) abundance measured by enzymatic analysis in LT2 control livers, LT2-MYC tumors, and tumors regressed for 72 h (n = 4 LT2 ctrl, n = 3 LT2-MYC tumor, n = 3 72 h reg tumor, data represented as univariate scatter plot with median, unpaired one-tailed t-test, LT2 ctrl versus LT2-MYC P = 0.01, LT2-MYC versus 72 h reg P = 0.14, LT2 ctrl versus 72 h reg P = 0.4, Bonferroni adjusted P-value is 0.02). Western blot analysis of HNRNPA1 in LT2-MYC tumors, control livers, and tumors regressed for 72 h (n = 3 each group). Source data are available online for this figure. Download figure Download PowerPoint Figure 3. GCLC is attenuated by miRNA-18a in MYC-driven liver tumors A. Western blot analysis of GCLC and MYC protein expression in conditional liver tumor cells derived from an LT2-MYC tumor (Western blot is representative of a minimum of four experimental replicates). B. Quantitative PCR (qPCR) analysis of miR-18a expression in LT2-MYC tumors, control liver tissues, and tumors regressed for 3 days (n = 4 each group, data represented as univariate scatter plot with median, unpaired two-tailed t-test, LT2 ctrl versus LT2-MYC tumor P = 0.002, LT2-MYC tumor versus 72 h regression tumors P = 0.001). C. qPCR analysis of miR-18a expression in conditional liver tumor cells treated with doxycycline (data represented as univariate scatter plot with median, data points represent three experimental replicates comprised of three technical replicates each, unpaired two-tailed t-test; compared to 0 h: 24 h, P = 0.14; 48 h, P = 0.008; 72 h, P = 0.005; 96 h, P = 0.003, **P-values for 48 h, 72 h, and 96 h fall below Bonferroni adjusted P-value of 0.01). D. Luciferase reporter expression in cultured murine liver tumor cells treated with a miR-18a mimic or control. Wt, wild-type Gclc 3′ UTR; mutated, Gclc 3′ UTR with four base pairs of the putative miR-18a binding site mutated (data represented as normalized mean ± SEM of three experimental replicates with three technical replicates each, unpaired two-tailed t-test, Wt UTR ctrl versus 18a mimic P = 0.0002, mutated UTR control versus 18a mimic P = 0.14). E. Western blot (WB) analysis of GCLC protein expression following treatment of cultured LT2-MYC liver tumor cells with locked nucleic acid (LNA) inhibitors of miR-18a (WB rep" @default.
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• W2590751720 title "<scp>MYC</scp> ‐driven inhibition of the glutamate‐cysteine ligase promotes glutathione depletion in liver cancer" @default.
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• W2590751720 doi "https://doi.org/10.15252/embr.201643068" @default.
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