Glutamine‐utilizing transaminases are a metabolic vulnerability of TAZ/YAP‐activated cancer cells

Scientific Report16 April 2018free access Transparent process Glutamine-utilizing transaminases are a metabolic vulnerability of TAZ/YAP-activated cancer cells Chih-Sheng Yang Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Eleni Stampouloglou Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Nathan M Kingston Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Liye Zhang Section of Computational Biomedicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Stefano Monti Section of Computational Biomedicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Xaralabos Varelas Corresponding Author [email protected] orcid.org/0000-0002-2882-4541 Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Chih-Sheng Yang Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Eleni Stampouloglou Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Nathan M Kingston Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Liye Zhang Section of Computational Biomedicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Stefano Monti Section of Computational Biomedicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Xaralabos Varelas Corresponding Author [email protected] orcid.org/0000-0002-2882-4541 Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA Search for more papers by this author Author Information Chih-Sheng Yang1,‡, Eleni Stampouloglou1,‡, Nathan M Kingston1,‡, Liye Zhang2,†, Stefano Monti2 and Xaralabos Varelas *,1 1Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA 2Section of Computational Biomedicine, Department of Medicine, Boston University School of Medicine, Boston, MA, USA †Present address: School of Life Science and Technology, ShanghaiTech University, Shanghai, China ‡These authors contributed equally to this work *Corresponding author. Tel: +1 617 358 4575; E-mail: [email protected] EMBO Rep (2018)19:e43577https://doi.org/10.15252/embr.201643577 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 The transcriptional regulators TAZ and YAP (TAZ/YAP) have emerged as pro-tumorigenic factors that drive many oncogenic traits, including induction of cell growth, resistance to cell death, and activation of processes that promote migration and invasion. Here, we report that TAZ/YAP reprogram cellular energetics to promote the dependence of breast cancer cell growth on exogenous glutamine. Rescue experiments with glutamine-derived metabolites suggest an essential role for glutamate and α-ketoglutarate (AKG) in TAZ/YAP-driven cell growth in the absence of glutamine. Analysis of enzymes that mediate the conversion of glutamate to AKG shows that TAZ/YAP induce glutamic–oxaloacetic transaminase (GOT1) and phosphoserine aminotransferase (PSAT1) expression and that TAZ/YAP activity positively correlates with transaminase expression in breast cancer patients. Notably, we find that the transaminase inhibitor aminooxyacetate (AOA) represses cell growth in a TAZ/YAP-dependent manner, identifying transamination as a potential vulnerable metabolic requirement for TAZ/YAP-driven breast cancer. Synopsis Elevated levels of the transcriptional regulators TAZ/YAP, key effectors of Hippo pathway signalling, mediate breast cancer cell growth dependence on exogenous glutamine. Cancer cells with high TAZ/YAP activity are sensitive to transaminase inhibition. High TAZ/YAP levels alter cellular energetics to promote breast cancer cell growth dependence on exogenous glutamine. TAZ/YAP promote the expression of glutamic–oxaloacetic transaminase (GOT1) and phosphoserine aminotransferase (PSAT1). Transaminase inhibition represses breast cancer cell growth in a TAZ/YAP dependent manner. Introduction Altered cellular energetics is an established hallmark of cancer 1, a premise supported by observations that oncogenes directly modulate metabolic circuits required for tumorigenesis 2-5. Oncogenic alterations in metabolic enzymes contribute to a variety of cancer-associated traits, including uncontrolled cell proliferation, alterations in cell polarity, increased metastatic ability, and evasion from cell death, indicating that aberrant cellular metabolism acts as a tumorigenic "driver" 2, 6-9. Cancer-specific metabolic reprogramming therefore represents an exciting avenue for the development of novel diagnostic tools and targeted cancer therapy 10, 11. The paralogous transcriptional regulators TAZ and YAP (herein referred to together as TAZ/YAP) have emerged as central factors in cancer biology. Nuclear TAZ/YAP activity has been shown to drive cell proliferation, survival, and mobility, and has key roles in directing cell fate 12. Increased levels and activity of TAZ/YAP have also been shown to correspond to high-grade tumors that generally lack effective therapeutics 12, 13. Advanced breast cancers in particular exhibit high nuclear TAZ/YAP levels and rely on nuclear TAZ/YAP transcriptional activity for driving breast cancer cell growth and aggressiveness 14, 15. Thus, in-depth understanding of the processes regulated by TAZ/YAP may provide important insight into the etiology of cancer and offer therapeutic opportunities. Here, we report that TAZ/YAP promote glutamine dependence in breast cancer cells and activate the expression of glutamine-utilizing transaminases to support cell growth. We found that glutamine deprivation greatly reduces the growth of breast cancer cells with high TAZ/YAP levels and that knockdown of TAZ/YAP mitigates cell death caused by glutamine depletion. Interestingly, we found that TAZ/YAP promote the expression of the transaminases GOT1 and PSAT1, and blockade of transamination with aminooxyacetate (AOA) suppresses the growth of breast cancer cells in a TAZ/YAP-dependent manner. Collectively, our data indicate that glutamine addiction of breast cancer cells is mediated by TAZ/YAP and suggest that targeting of transamination could be exploited for breast cancer therapies to attenuate TAZ/YAP-driven tumor growth. Results and Discussion TAZ/YAP control glutamine dependency of breast cancer cells Given the emerging oncogenic roles of TAZ/YAP 12, 13, we thought to analyze the nutrient requirements associated with TAZ/YAP activation to uncover susceptible metabolic processes for potential targeting of cancer cells with hyper-activated TAZ/YAP. To this end, we identified gene expression changes resulting from siRNA-mediated depletion of TAZ/YAP in MDA-MB-231 breast cancer cells (data from Enzo et al 16), which is a cell line that exhibits high nuclear TAZ/YAP activity 17, and performed Gene Set Enrichment Analysis (GSEA) to determine whether any relationships exist with gene sets representing distinct metabolic features 8 (Dataset EV1). Among 38 molecular signatures examined, 13 were downregulated upon TAZ/YAP deficiency (Table EV1), suggesting a role for TAZ/YAP in stimulating these metabolic processes. Notably, the gene set representing amino acid metabolism showed the strongest statistical association with TAZ/YAP activity (Fig 1A and Table EV1). Figure 1. Breast cancer cell lines with elevated levels of TAZ/YAP cancer exhibit glutamine dependence A. GSEA shows enrichment of amino acid metabolism-associated genes in gene expression signatures induced by TAZ/YAP. B. Lysates were isolated from density-matched mammary cells cultured in complete medium and examined for endogenous TAZ/YAP protein levels by immunoblotting. GAPDH levels were used to control loading. C. Cell growth of a panel of human breast cancer cell lines and a non-malignant human mammary epithelial cell line (HMEC) HMT-3522 S1 was measured in glutamine (Q)-free medium and then normalized to growth in complete medium. The reduction in culture size following glutamine starvation is defined as glutamine-dependent growth (red), while the remaining growth following glutamine starvation is defined as glutamine-independent growth (blue). The average growth from three independent experiments is shown (±SD). D, E. For the indicated cell lines, glutamine dependence measured in (C) was plotted against the expression levels of TAZ/YAP measured in (B) (shown in D), or the relative expression levels of the TAZ/YAP targets CTGF/CYR61/ANKRD1/EDN1 (determined from data available from the CCLE) (shown in E) to examine the correlation (A.U., arbitrary units) between these two biological features (see Materials and Methods for details). Download figure Download PowerPoint Targeting amino acid metabolic enzymes in cancer cells has shown promise as a therapeutic strategy. In particular, enzymes important for metabolizing the "non-essential" amino acid glutamine have emerged as important mediators of cancer cell growth (i.e., proliferation and survival) 10, 11, 18-21, particularly in aggressive breast cancer cells 21. We observed that several genes that encode important regulators of glutamine metabolism were reduced in expression following TAZ/YAP knockdown in MDA-MB-231 cells (Fig EV1), which encouraged us to test the importance of glutamine in TAZ/YAP expressing cells. To start, we tested whether TAZ/YAP levels correlate with glutamine dependence in a panel of human mammary cells, including eight breast cancer cell lines and a non-malignant human mammary epithelial cell (HMEC) line HMT-3522 S1 22. Immunoblotting for TAZ and YAP showed variable protein levels among these cells lines, ranging from very high levels in the more aggressive breast cancer cells (such as in MDA-MB-231 and HCC38) to very low levels in normal mammary epithelial cells (HMT-3522 S1; Fig 1B). The removal of glutamine from culture medium revealed that many cells exhibited glutamine-dependent growth (Fig 1C, red bars), whereas others grew robustly in the absence of exogenous glutamine (Fig 1C, blue bars). A strong positive correlation was observed between TAZ/YAP levels and glutamine dependence across these cells, with the growth of cells with pronounced levels of TAZ/YAP showing very strong glutamine dependence (Fig 1D). By examining gene expression data available from the Cancer Cell Line Encyclopedia (CCLE) project 23, we also observed a strong positive correlation between glutamine dependence and the expression of the YAP/TAZ target genes CTGF, CYR61, ANKRD1, and EDN1 (Fig 1E). Taken together, these observations suggested that TAZ/YAP activity may alter metabolic processes that drive exogenous glutamine reliance in breast cancer cells. Click here to expand this figure. Figure EV1. The expression of several genes encoding regulators of glutamine metabolism is reduced following TAZ/YAP knockdownThe relative change in the expression of genes encoding glutamine regulators was examined in microarray data available from Enzo et al 16 in MDA-MB-231 cells following TAZ/YAP knockdown. Download figure Download PowerPoint Glutamine depletion has been shown to trigger cancer cell death 18, 19. To further examine the dependence on glutamine in cells with different TAZ/YAP levels, we monitored the growth of two "high" TAZ/YAP cell lines (MDA-MB-231 and HCC38) and two "low" TAZ/YAP cell lines (BT474 and HMT-3522 S1) in glutamine replete and depleted media. Cells were seeded at comparable numbers (Fig 2A, Day 0), and after a 4-day incubation in complete medium, cell confluency increased in all cells examined [Fig 2A, Day 0 vs. Day 4 + Glutamine (Q)]. Removal of glutamine slightly reduced the confluence of low TAZ/YAP cells compared to that in complete medium (Fig 2A, left panel, Day 4 ± Q). By contrast, only few cells with high levels of TAZ/YAP survived glutamine-free conditions as evidenced by a significant drop of cell numbers (Fig 2A, right panel, Day 0 vs. Day 4-Q), suggesting that glutamine deprivation induces the death of cells with high levels of TAZ/YAP. We then performed cell counting and trypan blue exclusion assays to directly monitor cell death in MDA-MB-231 and HCC38 cells transfected with control siRNA or siRNA targeting TAZ, YAP, or both TAZ and YAP together. The efficacies of the siRNAs were validated by immunoblotting for TAZ and YAP levels (Fig EV2). In glutamine-free medium, the numbers of both MDA-MB-231 and HCC38 cells that received control siRNA profoundly declined after being switched to glutamine-free medium (Fig 2B, black lines), which was accompanied by an increase in cell death (Fig 2C, black bars). Interestingly, TAZ/YAP deficiency prevented the decline of the cell population (Fig 2B, red lines) and protected both cell lines against cell death (Fig 2C, gray bars) caused by glutamine deprivation. TAZ depletion alone similarly protected the decline of the cell population (Fig 2B, blue lines) following glutamine deprivation, albeit at a lower level than depletion of both TAZ and YAP, whereas YAP depletion alone had minimal effects. This predominant reliance on TAZ for mediating glutamine dependence is consistent with a more dominant role for TAZ in mediating tumorigenic phenotypes and clinical outcomes in breast cancers 13. Figure 2. Downregulation of TAZ/YAP alleviates glutamine dependence of breast cancer cells A. HMT-3522 S1 and BT474 (low TAZ/YAP) as well as MDA-MB-231 and HCC38 (high TAZ/YAP) cells seeded at similar density were cultured in complete or Q-deprived conditions. Representative photographs were taken before (Day 0) and 4 days after media change (Day 4). Scale bars = 100 μm. B, C. Cells transfected with control siRNA (black line) or siRNA targeting either TAZ (blue line), YAP (green line), or both TAZ and YAP (red line) were cultured in glutamine-free medium. (B) Cell numbers and (C) cell death were monitored at the indicated time points after medium switch. The average from three independent experiments +SEM is shown (*P < 0.05; **P < 0.01; ***P < 0.001; unpaired two-tailed t-test). D, E. MDA-MB-231 cells were engineered to constitutively express mouse TAZ (mTAZ), which is insensitive to siRNA targeting human TAZ. Cells expressing mTAZ along with control cells were transfected with control siRNA or siRNA targeting TAZ/YAP, and then: (D) the levels of endogenous human TAZ, ectopically expressed mTAZ, and GAPDH (as a loading control) were examined by immunoblotting lysates from respective cell lines; or (E) were cultured in Q-deprived conditions, and the relative growth of the cells was determined as a percentage of growth relative to complete media. The average from three independent experiments +SEM is shown (**P < 0.01; unpaired two-tailed t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Efficacy of TAZ and YAP siRNAMDA-MB-231 or HCC38 cells were transfected with indicated siRNA. Lysates were analyzed by immunoblotting with a TAZ/YAP antibody (CST, #8418) to examine antibody specificity and TAZ and YAP knockdown efficiency. Download figure Download PowerPoint The more pronounced effects of TAZ depletion on cell growth following glutamine deprivation prompted us to next test the consequences of TAZ expression. For this, we generated MDA-MB-231 cells stably expressing Mus musculus TAZ (mTAZ) that is not targeted by the human siRNA we used for endogenous TAZ depletion (Fig 2D). We found that expression of mTAZ was sufficient to reverse the growth protective effects of TAZ/YAP depletion following glutamine withdrawal, leading to a marked decline in cell growth after being switched to glutamine-free medium, similar to what was observed in control cells normally expressing high levels of TAZ/YAP (Fig 2E). Together, these data implicate TAZ as an essential mediator of glutamine addiction of breast cancer cells, with YAP playing a redundant role. TAZ/YAP promote anaplerotic entry of glutamine through transamination Glutamine serves as a precursor to provide carbon and nitrogen for the biosynthesis of metabolites that are involved in cancer survival and proliferation 10 (Fig 3A). To evaluate how different glutamine-metabolizing pathways mediate the growth of cancer cells with high TAZ/YAP levels, we tested the ability of several glutamine-derived metabolites to rescue the growth of MDA-MB-231 and HCC38 cells under glutamine-deprived conditions. Supplement with dimethyl glutamic acid, a cell-permeable analog of glutamate, rescued the growth of both breast cancer cell lines in a dosage-dependent manner (Fig 3B, red bars), consistent with the role of glutamate being the predominant metabolic fate of glutamine in proliferating cells 10, 24. Glutamate has been shown to promote cancer cell growth by maintaining the TCA cycle as an anaplerotic substrate, balancing cell redox status though glutathione synthesis and/or providing non-essential amino acids for protein synthesis 10 (Fig 3A). Notably, the addition of dimethyl 2-oxoglutarate, a membrane permeable analog that elevates mitochondrial α-ketoglutarate (AKG) levels to promote the TCA cycle, greatly restored culture size in both cell lines following glutamine withdrawal (Fig 3B, blue bars). The addition of the glutathione precursor N-acetylcysteine (NAC) (Fig 3B, green bars) or non-essential amino acids (Fig 3C, purple bars) had limited effects. These results suggest that the TCA cycle is a main metabolic destiny of glutamine in supporting the growth of cancer cells expressing high levels of TAZ/YAP. Figure 3. TAZ/YAP are required for glutamine-utilizing transaminase expression in breast cancer cells A depiction of the metabolic fates of glutamine. MDA-MB-231 and HCC38 cells were cultured in glutamine starvation medium supplemented with indicated metabolites for 48 h. The addition of dimethyl glutamate and dimethyl 2-oxoglutarate (AKG) resumed cell growth in glutamine-free medium in a dosage-dependent manner, while the supplement of other glutamine-derived downstream metabolites showed no, or marginal effects. The average from three independent experiments +SEM is shown (*P < 0.05; **P < 0.01; ***P < 0.001; unpaired two-tailed t-test). The relative expression levels of 588 genes altered in expression by knockdown of TAZ/YAP (i.e., TAZ/YAP gene expression signature) were examined in gene expression data from 1,088 breast cancer biopsy samples curated in TCGA BRCA dataset and 112 paired normal samples. The analysis of this data is shown as a heat map with each row representing a TAZ/YAP-regulated gene and each column representing a sample that was ranked by TAZ/YAP activity score (purple bar on the top) calculated by the ASSIGN analysis 26. Breast cancer samples (red) and normal samples (black) separated into two clusters, with the cancer samples aligning with higher TAZ/YAP activity. TAZ/YAP activity positively correlated with the expression of several transaminases and anti-correlated with the expression of both isoforms of glutamate dehydrogenase in 1,200 mammary biopsy samples analyzed. The Spearman's correlation coefficient [R] between TAZ/YAP activity and indicated metabolic enzymes is shown by the length and color code of each bar. The corresponding P-values are shown next to each bar. MDA-MB-231 cells were transfected with control siRNA (siCTL) or siRNAs targeting TAZ and YAP (siTAZ + siYAP), or a separate siRNA targeting both TAZ and YAP (siTAZ/YAP), and the protein levels of the transaminases GOT1 and PSAT1, as well as GAPDH (as a loading control) were assessed by immunoblotting. The changes in GOT1 and PSAT1 protein levels from three separate experiments were quantified relative to GAPDH, and the average +SEM is shown (***P < 0.001; unpaired two-tailed t-test). RT–qPCR was performed to determine the relative expression of GOT1 and PSAT1 mRNA in MDA-MB-231 cells treated with control siRNA, siRNAs targeting TAZ and YAP, or a separate siRNA targeting both TAZ and YAP. The average expression levels from three independent experiments +SEM are shown (*P < 0.05; ***P < 0.001; unpaired two-tailed t-test). MDA-MB-231 cells were subjected to ChIP analysis using control rabbit IgG, TAZ, or TEAD4 antibodies. Samples were analyzed by RT–qPCR using primers recognizing an enhancer for the GOT1 gene, which is illustrated at the top of the panel. Normalized % input values are shown as the average of three independent experiments +SEM (*P < 0.05; **P < 0.01; unpaired two-tailed t-test). Control cells and cells ectopically expressing mTAZ or MYC were transfected with control siRNA or siRNA targeting TAZ/YAP, and then, the relative expression levels of GOT1, CTGF, MYC, YAP, and human TAZ mRNA were assessed by RT–qPCR. Note the significant rescue of GOT1 and CTGF reduction resulting from TAZ/YAP depletion following the expression of mTAZ. The average from three independent experiments +SEM is shown (**P < 0.01; unpaired two-tailed t-test). Download figure Download PowerPoint Glutamate can be converted to AKG through transamination or oxidative deamination. The former is mediated by transaminases, including the glutamic–oxaloacetic transaminases GOT1 and GOT2 (GOT1/2), the glutamate pyruvate transaminases GPT1 and GPT2 (GPT1/2), and the phosphoserine aminotransferase PSAT1, while the latter is controlled by the glutamate dehydrogenases GLUD1 and GLUD2 (GLUD1/2). To gain more insights on how TAZ/YAP control this metabolic step in a pathophysiological setting, we analyzed gene expression profiles from 1,088 breast cancer biopsies available from The Cancer Genome Atlas (TCGA) BRCA dataset 25, and compared them to 112 paired normal samples for statistical associations between TAZ/YAP activity and the expression levels of aforementioned enzymes. To this end, we examined the expression pattern of a group of TAZ/YAP target genes as a proxy for TAZ/YAP activity and performed Adaptive Signature Selection and InteGratioN (ASSIGN) analysis to calculate the TAZ/YAP activity score for each biopsy sample (See Shen et al 26 and Materials and Methods). When ranked by their TAZ/YAP activity score (Fig 3C, purple bars), most tumor samples (Fig 3C, red bars) were clustered together and showed a higher score than their surrounding benign tissues (Fig 3C, black bars), consistent with reported elevation of TAZ/YAP activity in breast cancer 14, 17. Similar to what previous studies have suggested 14, 27, 28, basal and triple-negative breast cancer samples exhibited higher TAZ/YAP activity than other subtypes (Fig EV3). Interestingly, by examining the correlation of TAZ/YAP activity with genes encoding glutamine metabolism regulators, we found a strong positive correlation with GOT1 and PSAT1 expression and a negative correlation with glutamate dehydrogenase (GLUD1/2; Fig 3D), suggesting that TAZ/YAP promote transamination over oxidative deamination. Click here to expand this figure. Figure EV3. TAZ/YAP activity in breast cancer subtypesPatient samples curated in TCGA were classified into different subtypes by PAM50. ASSIGN analysis was performed (see Materials and Methods) to calculate TAZ/YAP activity score and is shown as a box plot in each subtype. ANOVA showed a significant difference in TAZ/YAP activity scores between tumor subtypes (P < 2.2e-16 for both classification methods). The Mann–Whitney U-test was performed to evaluate whether triple-negative breast cancer samples have a higher TAZ/YAP ASSIGN score compared with ER/Her2 samples, and whether basal breast cancer samples have a higher TAZ/YAP ASSIGN score compared with other subtypes. In both cases, triple-negative and basal breast cancer samples show significantly higher TAZ/YAP activity (P < 2.2e-16). Download figure Download PowerPoint Given the established oncogenic roles of GOT1 and PSAT1 in breast cancer and their strong associations with TAZ/YAP activity in our ASSIGN analysis, we investigated whether TAZ/YAP control the levels of these transaminases. For this, we examined GOT1 and PSAT1 mRNA levels by quantitative real-time PCR (qPCR) or protein levels by immunoblotting in cell lysates isolated from MDA-MB-231 cells transfected with control siRNA or two different sets of siRNA targeting TAZ and YAP. We found that depletion of TAZ/YAP reduced both the protein (Fig 3E) and mRNA (Fig 3F) levels of GOT1 and PSAT1. To investigate whether TAZ/YAP directly mediate the control of GOT1 and PSAT1 gene expression, we analyzed recent chromatin conformation capture (3C) and chromatin immunoprecipitation (ChIP)-sequencing data for YAP/TAZ from MDA-MB-231 cells 29 searching for binding to promoter or enhancer regions associated with these genes. We identified a potential binding site for YAP/TAZ and the TEAD transcription factors, which are binding partners of YAP/TAZ that are known to direct the pro-tumorigenic functions of YAP/TAZ 30, in a GOT1 enhancer (Fig 3G). Binding sites associated with the PSAT1 gene were less clear. The potential association with a GOT1 enhancer prompted us to perform our own ChIP experiment using TAZ and TEAD antibodies, which revealed that indeed TAZ and the TEAD transcription factors are bound to the GOT1 enhancer (Fig 3G). Given the direct association of TAZ with the GOT1 gene, we next investigated whether TAZ expression was sufficient to induce GOT1 expression. For this, we used the MDA-MB-231 cells we generated that stably express mTAZ (Fig EV4), which is not targeted by the siRNA that recognizes endogenous human TAZ. We found that expression of mTAZ was sufficient to increase the expression of GOT1 following the knockdown of human TAZ/YAP, which was similar to what we observed for the TAZ/YAP target CTGF (Fig 3H). GOT1 expression is known to be affected by the levels of the transcription factor MYC 31, an important mediator of glutamine metabolism in cancer cells 32. Given the association between MYC and TAZ/YAP activity in directing cell growth 33, we considered the possibility that changes in MYC levels contribute to the reduced levels of GOT1 following TAZ/YAP knockdown in MDA-MB-231 cells. To investigate the contribution of MYC to GOT1 expression, we generated MDA-MB-231 cells stably expressing MYC (Fig EV4). MYC expression was insufficient to rescue the reduced expression of GOT1 following TAZ/YAP knockdown (Fig 3I), suggesting that TAZ/YAP directly induce the expression of GOT1. We did, however, observe changes in MYC mRNA and protein levels following TAZ/YAP knockdown, even for MYC expressed from a CMV promoter (Fig 3H), indicating that TAZ/YAP control MYC levels. Given the importance of MYC in controlling glutamine metabolism and dependence 32, it is thus likely that TAZ/YAP-mediated control of MYC levels contributes to the metabolic phenotypes observed in breast cancer cells with high TAZ/YAP levels. Click here to expand this figure. Figure EV4. MDA-MB-231 cells expressing MYC or mTAZMDA-MB-231 cells were engineered to constitutively express mouse TAZ (mTAZ), which is insensitive to siRNA targeting human TAZ. Control cells and cells expressing MYC or mTAZ were transfected with control siRNA or siRNA targeting TAZ/YAP, and the levels of endogenous human TAZ and ectopically expressed mTAZ, MYC, and GAPDH (as a loading control) were examined by immunoblotting lysates from respective cell lines. Download figure Download PowerPoint Inhibition of transamination preferentially suppresses breast cancer cells expressing high levels of TAZ/YAP Given that glutamine-utilizing transaminases have been shown to support cell proliferation and stem cell renewal 20, 34, we hypothesized that elevated levels of these enzymes might play key roles in cancer cells with high TAZ/YAP activity. To test this idea, we examined how the glutamate-dependent transaminase inhibitor AOA modulates the growth of cells expressing high (MDA-MB-231 and HCC38) or low levels of TAZ/YAP (BT474 and HMT-3522 S1). The growth of cells expressing "low" TAZ/YAP levels was refractory to the inhibition of transamination, indicating their growth was mostly independent of this metabolic reaction (Fig 4A). By contrast, the growth of two "high" TAZ/YAP cells was profoundly suppressed by AOA, resulting in more than 60% reduction in relative growth at highest dose tested (Fig 4A). Notably, the growth deficiency in the "high" TAZ/YAP MDA-MB-231 cells following AOA treatment was partially rescued by increased levels of exogenous AKG or aspartate (Fig EV5), consistent with a central role of the GOT transaminases in these cells, as suggested by prior observations 31. Figure 4. Blockade of transamination suppresses the growth of breast cancer cells in a TAZ/YAP-dependent manner The indicated "TAZ/YAP-low" and "TAZ/YAP-high" cells were treated with AOA (0, 0.25, 0.5, 1.0 mM) in complete medium. The culture size of AOA- and mock-treated cells was examined 48 h post-treatment, and the relative growth of AOA/mock was determined as a percentage. Notably, the breast cancer cells expressing elevated levels of TAZ/YAP were more sensitive to AOA. The average