Abstract
Metabolic reprogramming by oncogenic signals promotes cancer initiation and progression. The oncogene KRAS and tumour suppressor STK11, which encodes the kinase LKB1, regulate metabolism and are frequently mutated in non-small-cell lung cancer (NSCLC). Concurrent occurrence of oncogenic KRAS and loss of LKB1 (KL) in cells specifies aggressive oncological behaviour1,2. Here we show that human KL cells and tumours share metabolomic signatures of perturbed nitrogen handling. KL cells express the urea cycle enzyme carbamoyl phosphate synthetase-1 (CPS1), which produces carbamoyl phosphate in the mitochondria from ammonia and bicarbonate, initiating nitrogen disposal. Transcription of CPS1 is suppressed by LKB1 through AMPK, and CPS1 expression correlates inversely with LKB1 in human NSCLC. Silencing CPS1 in KL cells induces cell death and reduces tumour growth. Notably, cell death results from pyrimidine depletion rather than ammonia toxicity, as CPS1 enables an unconventional pathway of nitrogen flow from ammonia into pyrimidines. CPS1 loss reduces the pyrimidine to purine ratio, compromises S-phase progression and induces DNA-polymerase stalling and DNA damage. Exogenous pyrimidines reverse DNA damage and rescue growth. The data indicate that the KL oncological genotype imposes a metabolic vulnerability related to a dependence on a cross-compartmental pathway of pyrimidine metabolism in an aggressive subset of NSCLC.
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Acknowledgements
We thank A. Jaffe and members of the DeBerardinis laboratory for critiquing the manuscript and J. Kozlitina for statistical expertise. R.J.D. is supported by grants from the NIH (R01CA157996), Cancer Prevention and Research Institute of Texas (CPRIT RP130272), Robert A. Welch Foundation (I1733) and H.H.M.I. (Faculty Scholars Program). J.K. is supported by an American Lung Association Senior Research Training Fellowship (RT-306212). D.H.C. is supported by NIH grant (1R01CA196912). J.D.M., J.R.C., P.V. and I.W. are supported by the University of Texas Lung Specialized Programs of Research Excellence (SPORE) grant (P50CA70907). J.D.M. is also supported by NIH grant CA176284 and CPRIT grants RP120732 and RP110708.
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J.K. and R.J.D. designed the study and wrote the paper. Z.H. performed the metabolomics. L.C. and M.N. provided biostatistics expertise. E.C. provided advice about replication-fork stalling. K.L. and J.X. performed ChIP–qPCR and provided advice on epigenetics. E.W., J.V. and D.L. provided human NSCLC samples for metabolomics. K.U.-K. and L.G. provided expertise in metabolomics and transcript analysis. C.G.P., D.H.C., P.V., J.R.-C. and I.W. performed tumour microarrays. Y.-F.L. and B.P.C.C. performed DNA fibre assays. B.F. provided expertise on AMPK. D.B. performed transient gene silencing. L.A.B. and J.V.H. provided reverse-phase proteomics and patient survival data. K.E.H. and J.D.M. provided cell lines, gene expression data and intellectual input regarding molecular lung cancer subtypes.
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Extended data figures and tables
Extended Data Figure 1 Altered urea cycle metabolism in KL cells.
a, Illustration of the urea and tricarboxylic acid (TCA) cycle. Metabolic alterations mediated by concurrent mutations of KRAS and LKB1 render cells dependent on CPS1 for pyrimidine synthesis. Generally, mitochondrial and cytosolic carbamoyl phosphate are thought to follow distinct metabolic routes into the urea cycle and pyrimidine biosynthesis, respectively. In KL cells, however, CPS1 supports nucleotide homeostasis by providing an alternative supply of carbamoyl phosphate for de novo pyrimidine synthesis. Dependence on CPS1 is exacerbated by mutant KRAS, perhaps because of the effects of this oncogene on the metabolism of glutamine and other nutrients in the mitochondria. αKG, α-ketoglutaric acid; CP, carbamoyl phosphate. b, Distribution of mRNA abundance of urea cycle-related enzymes in 203 cell lines. Pink dots are cancer cell lines and blue dots are bronchial or small airway epithelial cell lines. Statistical significance was assessed using a two-tailed Student’s t-test. Abundance data are in Supplementary Table 6. c, Abundance of urea cycle intermediates in the same cell lines used in Fig. 1a. Individual data points are shown as mean ± s.d. for three (carbamoyl phosphate) or four (all others) independent cultures. Statistical significance for citrulline and carbamoyl phosphate was assessed using a Wilcoxon signed-rank test. Other data were assessed using two-tailed Student’s t-tests. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Extended Data Figure 2 Metabolomic profiling of KL and K cancer cells and human NSCLC.
Left, relative abundance of metabolites extracted from five KL (H157, A549, H460, H2122, H1355) and five K (Calu-1, Calu-6, H1373, H358, H441) cell lines. Peak areas of each metabolite were normalized by total ion count and the heat map displays the average value for each metabolite; n = 4 independent cultures for each cell line. Right, relative abundance of metabolites extracted from four KL human tumours (tumours 5-, 8-, 9- and 11-KL) and seven K human tumours (tumours 1-, 2-, 3-, 4-, 6-, 7- and 10-K). Peak areas of each metabolite were normalized by total ion count followed by mean normalization and the heat map displays the average value for each metabolite; n = 3 independent fragments for each tumour except tumour 2-K (n = 6), tumour 8-KL (n = 6) and tumour 1-K (n = 9). KL cell lines or tumours are indicated in red, K cell lines or tumours are indicated in blue. The colours in the heat map reflect a log2 scale.
Extended Data Figure 3 Metabolites differentiating between KL and K human NSCLC.
Metabolites differentiating K from KL human tumours have variable importance in the projection (VIP) scores >1.0. Metabolites related to nitrogen metabolism are highlighted in red. The relative abundance of each metabolite is shown in the colour bar, where red indicates increased and green indicates decreased abundance. *Metabolites that also discriminated between K and KL cell lines in Fig. 1a and Supplementary Table 2. ^Metabolites closely related to those discriminating between K and KL cell lines in Fig. 1a (for example, hypoxanthine and xanthine in the tumours are related to xanthosine in the cell lines).
Extended Data Figure 4 Nitrogen-related metabolic pathways in K and KL cells and inverse correlation between CPS1 and LKB1.
a, Abundance of NOS3 protein in a number of K and KL cell lines. b, Distribution of mRNA abundance for NOS1 and NOS2 among 203 cell lines. Complete datasets including quantitative mRNA abundance of these genes are available in Supplementary Table 6. c, NOS activity in K and KL cells. Free NO was monitored in three cell lines of each genotype. Data are the mean ± s.d. of three independent cultures. d, Effect of silencing ornithine decarboxylase (ODC), an enzyme involved in polyamine synthesis from ornithine, in K and KL cells. Cell growth was measured by DNA content using a Perkin Elmer Victor X3 plate reader. Data are the mean ± s.d. of six independent cultures. e, Pearson’s correlation coefficients (r) between CPS1 mRNA and 176 proteins in 94 lung cancer cell lines. The rank of the LKB1 protein is indicated. Dashed lines demarcate correlation coefficients at a nominal P = 0.05. f, Scoring of LKB1 and CPS1 expression in TMA samples. For this analysis, tumours were considered positive if any CPS1 or LKB1 staining was detected (that is, an H-score greater than or equal to 1, as described in Methods); otherwise staining was considered negative. g, CPS1 protein expression in TMA tumour samples of different clinical stages. h, Abundance of CPS1 and LKB1 protein in patient-derived NSCLC xenografts. All patient-derived NSCLC xenografts had oncogenic KRAS mutations. i, Kaplan−Meier plot associating CPS1 expression with reduced survival. In the TCGA lung adenocarcinoma cohort (TCGA LUAD provisional, n = 230), LKB1 mutation or loss was observed in 19% of patient tumours (n = 43). For CPS1, a z-score threshold of 2.0 was used to identify tumours with high levels of expression; this included 5.2% (n = 12) of tumours. There was no difference in overall survival in patients with LKB1 alterations (deletion or mutation) versus those without an LKB1 alteration (P = 0.88). By contrast, patients whose tumours expressed high levels of CPS1 mRNA had much shorter periods of overall survival compared to other patients (15.2 versus 45.3 months, P < 0.0001). The western blot and NOS activity assay were performed twice and the ODC silencing experiment was repeated three times or more. Statistical significance was assessed using a two-tailed Student’s t-test. NS, not significant.
Extended Data Figure 5 LKB1 suppresses CPS1 expression through AMPK.
a, Expression of urea cycle and related enzymes in control A549 cells (empty vector, EV) and cells expressing wild-type LKB1. Data are the mean ± s.d. of three independent cultures. b, Abundance of CPS1 and LKB1 protein in A549 cells transfected with an empty vector (EV) or wild-type LKB1 (LKB1 WT). CB was used as a loading control. c, Left, the effect of expressing wild-type LKB1 or mouse CPS1 (mCPS1), alone or together, on H460 cell proliferation. EV is the empty vector control. Data are the mean ± s.d. of six or more independent cultures. Right, abundance of CPS1 and LKB1 protein in H460 cells stably expressing the empty vector or mCPS1. d, Top, effects of LKB1 silencing on CPS1 mRNA expression in cells with oncogenic KRAS and wild-type LKB1 (K cells). Data are the mean ± s.d. of three or more independent cultures. Western blot shows the abundance of LKB1 protein in cells transfected with control siRNA or siRNA targeting LKB1 (siLKB1). Bottom, effects of the AMPK activator A769662 on CPS1 mRNA expression in K cells. Data are the mean ± s.d. of three or more independent cultures. Western blot shows the abundance of total and phosphorylated acetyl-CoA carboxylase (pAcc, S79) in cells treated with DMSO or A769662 (250 μM). e, Effects of A769662-mediated AMPK activation on CPS1 mRNA expression in KL cells. Data are the mean ± s.d. of three or more independent cultures. f, Effects of constitutively active (CA) AMPK on CPS1 mRNA expression in H2122 and H460 cells. Data are the mean ± s.d. of three or more independent cultures. g, Abundance of CPS1, pAcc and constitutively active AMPKα in H2122 and H460 cells transfected with an empty vector (EV) or constitutively active (CA) AMPKα. Actin was used as a loading control. h, Left, effects of AMPK silencing on CPS1 mRNA expression in A549 cells without (EV) or expressing wild-type LKB1. Data are the mean ± s.d. of three independent cultures. Right, abundance of CPS1, LKB1 and AMPK proteins in A549 cells transfected with control siRNA or siRNA targeting AMPK (siAMPK). Actin was used as a loading control. i, Left, effects of the mTOR inhibitor Torin 1 on CPS1 mRNA expression in KL cells. Data are the mean ± s.d. of four or more independent cultures. Right, abundance of CPS1, phosphorylated S6 (pS6) ribosomal protein and phosphorylated 4E-BP1 (p4E-BP1) in KL cells. Actin was used as a loading control. j, Left, effects of TSC1 and TSC2 (siTSC1/2) silencing on CPS1 mRNA expression in A549 and H460 cells. Data are the mean ± s.d. of three independent cultures. Right, abundance of CPS1, LKB1 and pS6 in A549 cells. CB was used as a loading control. In a, d, f, j, statistical significance was assessed using two-tailed Student’s t-tests. *P < 0.05, **P < 0.01, ***P < 0.001, n.s., not significant. In c, e, h, i, statistical significance was assessed using a one-way ANOVA followed by Tukey’s multiple comparisons test. In c, *P < 0.05 compared to EV and −LKB1; #P < 0.05 compared to EV and +LKB1. In e, *P < 0.05 compared to no treatment; #P < 0.05 compared to 12 h A769662 treatment. In h, *P < 0.05 compared to EV and control siRNA; #P < 0.05 compared to EV and siAMPK; †P < 0.05 compared to LKB1 and control siRNA. All experiments were repeated three times or more.
Extended Data Figure 6 LKB1 regulates CPS1 transcription through AMPK-mediated effects.
a, Chromatin signatures at the CPS1 locus in A549 cells. Promoter and enhancer sequences are shaded. Arrowheads indicate amplicons for control (C), promoter (P) and enhancer (E1, E2) regions for ChIP–qPCR in b and c. b, Chromatin occupancy of H3K27ac, RNAPII, H3K4me3, CREB1, FOXA1, TEAD4 and IgG (negative control) in KL (A549, H460) and in K (Calu-1, H1373) cells. Data are the mean ± s.d. of two independent cultures, each with two technical replicates (total n = 4). c, Chromatin occupancy of H3K27ac, RNAPII, H3K4me3, CREB1, FOXA1, TEAD4 and IgG in A549 and H460 cells treated with DMSO or 250 μM A769662. Data are the mean ± s.d. of three independent cultures, each with two technical replicates (total n = 6). d, Effects of CREB1, FOXA1 and TEAD4 silencing on CPS1 mRNA expression in A549 and H460 cells. Data are the mean ± s.d. of three or more replicates. e, Abundance of CPS1, CREB1, FOXA1 and TEAD4 in A549 cells. CB was used as a loading control. In b and c, statistical significance was assessed using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001, NS, not significant. In d, statistical significance was assessed using a one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05 compared to control. ChIP–qPCR in b was performed twice. All other experiments were repeated three times or more.
Extended Data Figure 7 CPS1 addiction in a subset of NSCLC cell lines.
a, Abundance of CPS1 protein in cell lines transfected with a control esiRNA or esiRNA directed against CPS1. b, Effect of CPS1 silencing on cell death in K and KL cells. Data are the mean ± s.d. of three independent cultures. c, Effect of CPS1 silencing on A549 cell viability. Data are the mean ± s.d. of six independent cultures. d, Effect of lentiCRISPR–Cas9-mediated knockout of CPS1 on viability in H2122 and H460 cells. CellTiter-Glo assays were performed on pools of CPS1 knockout cells without first isolating clones. Data are the mean ± s.d. of six independent cultures. e, Abundance of CPS1 protein in H2122 and H460 control cells (EV) and a pool of cells infected with lentiviral CRISPR V2-CPS1 (CPS1). Actin was used as a loading control. f, Effect of knocking out CPS1 on H460-EV and H460-LKB1-WT cells (n = 6). g, Abundance of CPS1 in H460 cells expressing shCPS1-1 (sh#1, top) and shCPS1-2 (sh#2, bottom) with or without Dox induction. shREN is a Dox-inducible control shRNA, and actin was used as a loading control. h, Top, effects of mouse CPS1 (mCPS1) expression on viability in H460 cells expressing shCPS1-2. Data are the mean ± s.d. of three independent cultures. Bottom, abundance of CPS1 protein in H460 cells expressing mCPS1 with or without shCPS1 induction. Actin was used as a loading control. i, TUNEL staining of tumour tissues. 4′,6-diamidino-2-phenylindole (DAPI) was used to stain DNA. Scale bars, 500 μm. In d, statistical significance was assessed using a two-tailed Student’s t-test. ****P < 0.0001. In f and h, statistical significance was assessed using a one-way ANOVA followed by Tukey’s multiple comparisons test. ***P < 0.001, ****P < 0.0001. Tissue TUNEL staining was performed once. Viability assay (f) was performed twice. All other experiments were repeated three times or more.
Extended Data Figure 8 CPS1 expression and xenograft growth.
a, b, Abundance of CPS1 protein in H460 (a) and H2122 (b) xenografts expressing shCPS1-1 (top) and shCPS1-2 (bottom) with or without Dox induction. shREN is a Dox-inducible control shRNA and actin is a loading control. c, Effect of Dox-induction of shCPS1-1 (left) and shCPS1-2 (right) on H2122 xenograft growth. Nude mice were injected subcutaneously with H2122 shCPS1 (shCPS1-1, shCPS1-2) cells and Dox (200 mg kg−1) was introduced one day later. Each group (n = 8) is presented as mean tumour volume ± s.e.m. Statistical significance was assessed using a two-way ANOVA. **P < 0.01, ****P < 0.0001. Experiments were performed once (n = 8 mice).
Extended Data Figure 9 CPS1 silencing results in pyrimidine depletion and DNA damage.
a, Ammonia release from H460 cells expressing shREN or shCPS1-1 (n = 3). b, Ammonia release from H460 cells expressing shREN or shCPS1-2 in the presence and absence of glucose (Glc). Glucose deprivation provides a positive control for enhanced ammonia release in cancer cells (see ‘Metabolic Assays’ in the Methods). Data are the mean ± s.d. of three independent cultures, each with three technical replicates (total n = 9). c, Abundance of total CAD, phosphorylated CAD (pCAD) and phosphorylated 4E-BP1 (p4E-BP1) in K, L and KL cells. Actin was used as a loading control. d, Left, CPS1, CAD, LKB1 and phosphorylated AMPK (pAMPK) abundance in H460 cells without (EV) or with LKB1 expression. Cells were transfected with siRNA targeting CAD or CPS1. Actin was used as a loading control. Right, abundance of CPS1 and CAD protein in wild-type or CPS1-knockout H460 cells. Actin was used as a loading control. e, Relative abundance of pyrimidines and purines during expression of shREN (closed bars) or shCPS1-1 (open bars) (n = 6). f, Effects of CPS1 silencing on BrdU incorporation (n ≥ 3). DNA content and BrdU incorporation were assessed by flow cytometry following dual staining with BrdU and propidium iodide. g, Effect of CPS1 silencing on cell cycle distribution (n = 4). h, Abundance of CPS1 protein and γH2AX in H460-shREN and -shCPS1-1 cells with or without Dox induction. i, γH2AX in H460 xenografts. DAPI was used to stain DNA. Scale bars, 40 μm. j, Effects of CPS1 silencing on DNA track length measured by iododeoxyuridine (IdU) and chlorodeoxyuridine (CldU) incorporation. At least 104 tracks were measured for each condition. Scale bars, 2 μm. In b, statistical significance was assessed using a one-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05 compared to shREN with glucose; #P < 0.05 compared to shCPS1 with glucose (Glc). In a, e, g, j, statistical significance was assessed using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001. Tissue staining was performed once. Nucleotide measurements and DNA fibre assays were performed twice. All other experiments were repeated three times or more.
Extended Data Figure 10 Regulation of CAD transcription is distinct from that of CPS1 and pyrimidine nucleosides rescue DNA damage and proliferation of CPS1 silenced cells.
a, Chromatin signatures at the CAD locus in A549 cells. Promoter sequences are shaded. Arrowheads indicate amplicons for promoter (P1, P2) regions for ChIP–qPCR in b. b, Chromatin occupancy of H3K27ac, RNAPII, FOXA1, H3K4me3, CREB1, TEAD4 and IgG (negative control) in KL (A549, H460) and in K (Calu-1, H1373) cells. Data are the mean ± s.d. of two independent cultures, each with two technical replicates (total n = 4). c, Effects of CREB1, FOXA1 and TEAD4 silencing on CAD mRNA expression in A549 cells. Data are the mean ± s.d. of three independent cultures, each with two technical replicates (total n = 6). d, Effects of CREB1, FOXA1 and TEAD4 silencing on CAD protein abundance in A549 cells. CB was used as a loading control. e, The effect of supplementing culture medium with uridine and thymidine (UT) or adenosine (A) (100 μM final concentration) on γH2AX abundance in Dox-induced H460 cells expressing shREN or shCPS1-1. f, The effect of supplementing culture medium with uridine and thymidine or adenosine on anchorage-independent colony formation of H460 cells expressing shCPS1-1 (n = 3). The time point is after 20 days of Dox induction. g, Effects of nucleoside supplementation on proliferation of H460 cells expressing shCPS1-2. Data are the mean ± s.d. of three independent cultures, each with three technical replicates (total n = 9). h, Colonies from Fig. 4f. 1–4: shREN, 5–8: shCPS1-2; 1, 5, no treatment; 2, 6, Dox treatment; 3, 7, Dox and uridine and thymidine; 4, 8, Dox and adenosine. i, The effect of supplementing with uridine alone on anchorage-independent colony formation of H460 cells expressing shCPS1-2. Data are the mean ± s.d. of three independent cultures. j, Growth of subcutaneous H460 shREN-derived xenografts in presence and absence of Dox, with or without cisplatin. Mean tumour volume ± s.e.m. are shown for each group (n = 4) k, Growth of subcutaneous H2122 shCPS1-#2-derived xenografts in nude mice in the presence and absence of Dox (200 mg kg−1) introduced one day after implantation with or without cisplatin treatment (intraperitoneal injection at 2 mg kg−1 for 5–6 doses). Mean tumour volume ± s.e.m. are shown for each group (n = 4). In b, i, statistical significance was assessed using a two-tailed Student’s t-test. *P < 0.05, **P < 0.01, NS, not significant. In f, g, statistical significance was assessed using a one-way ANOVA followed by Tukey’s multiple comparisons test. In j, k, statistical significance was assessed using a two-way ANOVA followed by Tukey’s multiple comparisons test. In f, *P < 0.05 compared to shREN; #P < 0.05 compared to shCPS1; †P < 0.05 compared to shCPS1 and adenosine. In g, first four bars: *P < 0.05 compared to shREN without Dox; second four bars: *P < 0.05 compared to shCPS1 without Dox; #P < 0.05 compared to shCPS1 with Dox; †P < 0.05 compared to shCPS1 with Dox and uridine and thymidine. In j, k, *P < 0.05 compared to −Dox and −Cis; #P < 0.05 compared to −Dox and +Cis; †P < 0.05 compared to +Dox and −Cis. Xenograft experiments were performed once, ChIP–qPCR was performed twice and all other experiments were performed three times or more.
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Kim, J., Hu, Z., Cai, L. et al. CPS1 maintains pyrimidine pools and DNA synthesis in KRAS/LKB1-mutant lung cancer cells. Nature 546, 168–172 (2017). https://doi.org/10.1038/nature22359
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DOI: https://doi.org/10.1038/nature22359
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