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• W2913854500 abstract "•Cell sorting and metabolic tracing reveals cell-cycle-associated metabolites•Ornithine synthesis peaks during SG2M in transformed but not in normal cells•Cancer cells synthesize ornithine using ARG2 only, while normal cells use OAT•Knockdown of ARG2 suppresses cancer cell growth without compensation by OAT Alterations in cell-cycle regulation and cellular metabolism are associated with cancer transformation, and enzymes active in the committed cell-cycle phase may represent vulnerabilities of cancer cells. Here, we map metabolic events in the G1 and SG2M phases by combining cell sorting with mass spectrometry-based isotope tracing, revealing hundreds of cell-cycle-associated metabolites. In particular, arginine uptake and ornithine synthesis are active during SG2M in transformed but not in normal cells, with the mitochondrial arginase 2 (ARG2) enzyme as a potential mechanism. While cancer cells exclusively use ARG2, normal epithelial cells synthesize ornithine via ornithine aminotransferase (OAT). Knockdown of ARG2 markedly reduces cancer cell growth and causes G2M arrest, while not inducing compensation via OAT. In human tumors, ARG2 is highly expressed in specific tumor types, including basal-like breast tumors. This study sheds light on the interplay between metabolism and cell cycle and identifies ARG2 as a potential metabolic target. Alterations in cell-cycle regulation and cellular metabolism are associated with cancer transformation, and enzymes active in the committed cell-cycle phase may represent vulnerabilities of cancer cells. Here, we map metabolic events in the G1 and SG2M phases by combining cell sorting with mass spectrometry-based isotope tracing, revealing hundreds of cell-cycle-associated metabolites. In particular, arginine uptake and ornithine synthesis are active during SG2M in transformed but not in normal cells, with the mitochondrial arginase 2 (ARG2) enzyme as a potential mechanism. While cancer cells exclusively use ARG2, normal epithelial cells synthesize ornithine via ornithine aminotransferase (OAT). Knockdown of ARG2 markedly reduces cancer cell growth and causes G2M arrest, while not inducing compensation via OAT. In human tumors, ARG2 is highly expressed in specific tumor types, including basal-like breast tumors. This study sheds light on the interplay between metabolism and cell cycle and identifies ARG2 as a potential metabolic target. The cell cycle is of fundamental importance in cell biology and in biomedicine, as cancer, inflammation, and autoimmune disorders all involve cell proliferation. Mechanisms in the “committed” SG2M phase of the cell cycle, between onset of DNA synthesis (S) and mitosis (M), are commonly targeted by antiproliferative drugs (Chabner and Roberts, 2005Chabner B.A. Roberts Jr., T.G. Timeline: chemotherapy and the war on cancer.Nat. Rev. Cancer. 2005; 5: 65-72Crossref PubMed Scopus (1409) Google Scholar), which are highly effective but generally cause adverse side effects in normal proliferating tissues. On the other hand, cancer transformation is associated with altered metabolism (Hammoudi et al., 2011Hammoudi N. Ahmed K.B. Garcia-Prieto C. Huang P. Metabolic alterations in cancer cells and therapeutic implications.Chin. J. Cancer. 2011; 30: 508-525Crossref PubMed Scopus (71) Google Scholar) and expression of metabolic enzymes not commonly found in normal cells (Mazurek et al., 2005Mazurek S. Boschek C.B. Hugo F. Eigenbrodt E. Pyruvate kinase type M2 and its role in tumor growth and spreading.Semin. Cancer Biol. 2005; 15: 300-308Crossref PubMed Scopus (647) Google Scholar), which might be targeted with better specificity (Vander Heiden, 2011Vander Heiden M.G. Targeting cancer metabolism: a therapeutic window opens.Nat. Rev. Drug Discov. 2011; 10: 671-684Crossref PubMed Scopus (1079) Google Scholar). A combination of these two approaches, seeking cancer-associated metabolic enzymes active in the committed SG2M phase of the cell cycle, might identify new antiproliferative drug targets that are both effective and specific. Proliferating cells must synthesize a multitude of cellular components while simultaneously catabolizing nutrients to obtain energy, all while maintaining essential cell functions. How cells orchestrate these complex metabolic processes while progressing through the cell cycle is still poorly understood. In the yeast S. cerevisiae, respiration and redox state can undergo spontaneous cycles (Tu et al., 2005Tu B.P. Kudlicki A. Rowicka M. McKnight S.L. Logic of the yeast metabolic cycle: temporal compartmentalization of cellular processes.Science. 2005; 310: 1152-1158Crossref PubMed Scopus (691) Google Scholar) that can be coupled to the cell cycle (Papagiannakis et al., 2017Papagiannakis A. Niebel B. Wit E.C. Heinemann M. Autonomous metabolic oscillations robustly gate the early and late cell cycle.Mol. Cell. 2017; 65: 285-295Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar), and carbohydrate utilization occurs during the SG2M phase (Ewald et al., 2016Ewald J.C. Kuehne A. Zamboni N. Skotheim J.M. The yeast cyclin-dependent kinase routes carbon fluxes to fuel cell cycle progression.Mol. Cell. 2016; 62: 532-545Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). In mammalian cells, data obtained using synchronization methods indicate that central metabolic processes like glycolysis (Colombo et al., 2011Colombo S.L. Palacios-Callender M. Frakich N. Carcamo S. Kovacs I. Tudzarova S. Moncada S. Molecular basis for the differential use of glucose and glutamine in cell proliferation as revealed by synchronized HeLa cells.Proc. Natl. Acad. Sci. USA. 2011; 108: 21069-21074Crossref PubMed Scopus (124) Google Scholar), glycogen utilization (Favaro et al., 2012Favaro E. Bensaad K. Chong M.G. Tennant D.A. Ferguson D.J. Snell C. Steers G. Turley H. Li J.L. Günther U.L. et al.Glucose utilization via glycogen phosphorylase sustains proliferation and prevents premature senescence in cancer cells.Cell Metab. 2012; 16: 751-764Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar), glutamine catabolism (Ahn et al., 2017Ahn E. Kumar P. Mukha D. Tzur A. Shlomi T. Temporal fluxomics reveals oscillations in TCA cycle flux throughout the mammalian cell cycle.Mol. Syst. Biol. 2017; 13: 953Crossref PubMed Scopus (41) Google Scholar), and polyamine synthesis (Bettuzzi et al., 1999Bettuzzi S. Davalli P. Astancolle S. Pinna C. Roncaglia R. Boraldi F. Tiozzo R. Sharrard M. Corti A. Coordinate changes of polyamine metabolism regulatory proteins during the cell cycle of normal human dermal fibroblasts.FEBS Lett. 1999; 446: 18-22Crossref PubMed Scopus (52) Google Scholar) are cyclic. However, synchronization methods are problematic in that they block cell-cycle progression by inhibiting metabolic processes such as thymidine or mevalonate synthesis (Whitfield et al., 2002Whitfield M.L. Sherlock G. Saldanha A.J. Murray J.I. Ball C.A. Alexander K.E. Matese J.C. Perou C.M. Hurt M.M. Brown P.O. Botstein D. Identification of genes periodically expressed in the human cell cycle and their expression in tumors.Mol. Biol. Cell. 2002; 13: 1977-2000Crossref PubMed Scopus (1188) Google Scholar, Keyomarsi, 1996Keyomarsi K. Synchronization of mammalian cells by Lovastatin.Methods Cell Sci. 1996; 18: 109-114Crossref Scopus (5) Google Scholar), and hence may disturb metabolism. Moreover, most mammalian cells are difficult to synchronize (Cooper, 2004Cooper S. Rejoinder: whole-culture synchronization cannot, and does not, synchronize cells.Trends Biotechnol. 2004; 22: 274-276Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar), and consequently most data have been gathered from a few transformed cell lines that are amenable to synchronization, including HeLa and mouse 3T3 cells. This is problematic, since cell-cycle regulation clearly differs across cell types (Kung et al., 1990Kung A.L. Sherwood S.W. Schimke R.T. Cell line-specific differences in the control of cell cycle progression in the absence of mitosis.Proc. Natl. Acad. Sci. USA. 1990; 87: 9553-9557Crossref PubMed Scopus (213) Google Scholar), and it is of particular interest to study differences between normal and transformed cells. Clearly, systematic studies of metabolic events in the cell cycle across multiple cell types, including normal cells, are needed. To address this problem, we here report a systematic mapping of metabolic events in the G1 and SG2M stages in transformed and normal human cells, utilizing a combination of cell sorting and mass spectrometry-based isotope tracing. This approach does not require synchronization and is potentially applicable to any cell type. In the present study, this technique revealed that ornithine synthesis by mitochondrial arginase 2 (ARG2) is important in the committed phase of transformed, but not normal cells, representing a potential metabolic vulnerability of cancer cells. To enable the study of metabolic events in the cell cycle, we sought a method for separating cells into the G1 and “committed” SG2M phases that is applicable to any cell type and has minimal disturbance on cellular metabolism. We chose to sort cells by DNA content into 2n and 4n fractions, corresponding to the G1 and SG2M phases respectively, using the DNA-binding fluorescent molecule Hoechst-34580 (Figure 1A). This method provided robust quantification of DNA content in live cells with only 15-min incubation, and yet was nontoxic for up to 17 h in most cell types (Figures S1A–S1G). To obtain pure populations, we excluded cells in a window between 2n and 4n that contained G1/S-transition cells, as demonstrated by Hoechst-34580 versus geminin expression (Figure 1B). With this strategy, the markers Cdt1 for G1 and cyclin A for SG2M were exclusively found in the 2n and 4n fractions, respectively (Figure 1C), indicating high purity of the sorted fractions. We then analyzed 80,000 cells from the 2n and 4n fractions using liquid chromatography-mass spectrometry (LC-MS). Although cell sorting has been reported to distort cell redox state in astrocytes (Llufrio et al., 2018Llufrio E.M. Wang L. Naser F.J. Patti G.J. Sorting cells alters their redox state and cellular metabolome.Redox Biol. 2018; 16: 381-387Crossref PubMed Scopus (88) Google Scholar), and we cannot entirely exclude such effects, we found that redox couples were not altered in a concerted manner (Figures S1H–S1J). Also, to reduce artifacts caused by the sorting procedure, we throughout compare peak areas in a paired fashion, between fractions sorted from the same sample, which have been affected in the same way. Reassuringly, we observed that deoxyribonucleotide triphosphates (dNTPs), which are synthesized only during S phase (Bray and Brent, 1972Bray G. Brent T.P. Deoxyribonucleoside 5-triphosphate pool fluctuations during the mammalian cell cycle.Biochim. Biophys. Acta. 1972; 269: 184-191Crossref PubMed Scopus (69) Google Scholar, Skoog et al., 1973Skoog K.L. Nordenskjöld B.A. Bjursell K.G. Deoxyribonucleoside-triphosphate pools and DNA synthesis in synchronized hamster cells.Eur. J. Biochem. 1973; 33: 428-432Crossref PubMed Scopus (100) Google Scholar), were exclusively found in the 4n fraction (Figure 1D). To compare the cell-sorting approach with commonly used synchronization methods, we generated LC-MS data from HeLa cells synchronized in S phase using the double thymidine block (DTB) technique (Whitfield et al., 2002Whitfield M.L. Sherlock G. Saldanha A.J. Murray J.I. Ball C.A. Alexander K.E. Matese J.C. Perou C.M. Hurt M.M. Brown P.O. Botstein D. Identification of genes periodically expressed in the human cell cycle and their expression in tumors.Mol. Biol. Cell. 2002; 13: 1977-2000Crossref PubMed Scopus (1188) Google Scholar), and in G1 phase by lovastatin (Keyomarsi, 1996Keyomarsi K. Synchronization of mammalian cells by Lovastatin.Methods Cell Sci. 1996; 18: 109-114Crossref Scopus (5) Google Scholar). Although HeLa cells are among the easiest to synchronize, perfect synchrony is never attained, and in this case 20% of DTB cells were not in S phase, while 22% of lovastatin-treated cells were not in G1 (Figure S1K). Accordingly, we detected dNTPs also in lovastatin-synchronized cells (Figure 1E). Such cross-contaminations suggest that metabolite fold changes will be underestimated from synchronized populations, while the higher purity attained by cell sorting should give more power to detect cycling metabolites. We also noted a clear increase in the DNA damage marker ADP-ribose (Berger, 1985Berger N.A. Poly(ADP-ribose) in the cellular response to DNA damage.Radiat. Res. 1985; 101: 4-15Crossref PubMed Scopus (679) Google Scholar) in DTB-synchronized cells, but not in sorted SG2M cells (Figure 1F), consistent with reports that DTB can cause DNA damage (Kurose et al., 2006Kurose A. Tanaka T. Huang X. Traganos F. Darzynkiewicz Z. Synchronization in the cell cycle by inhibitors of DNA replication induces histone H2AX phosphorylation: an indication of DNA damage.Cell Prolif. 2006; 39: 231-240Crossref PubMed Scopus (55) Google Scholar). In addition, ribose-5-phosphate (Figure 1G) and the pentose phosphate pathway metabolite sedoheptulose-7-phosphate (Figure 1H) were markedly elevated in DTB-synchronized, but not in sorted SG2M cells, possibly indicating a disturbance in ribose metabolism. Taken together, these data indicate that our approach reliably detects cellular metabolites present in specific cell-cycle phases. To determine activities of enzymes and pathways in the G1 and SG2M phases, cells were pulse-labeled with a medium where glucose and all amino acids were fully 13C (Grankvist et al., 2018Grankvist N. Watrous J.D. Lagerborg K.A. Lyutvinskiy Y. Jain M. Nilsson R. Profiling the metabolism of human cells by deep 13C labeling.Cell Chem. Biol. 2018; 25: 1419-1427.e4Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar), followed by cell sorting as above (Figure 2A). Since metabolites in any given cell are 13C-labeled according to its metabolic activities during the 13C pulse, this design reveals cell-cycle-associated metabolic events as they occurred in the undisturbed culture, prior to cell sorting, and also reduces the impact of disturbances from the sorting procedure (Roci et al., 2016Roci I. Gallart-Ayala H. Schmidt A. Watrous J. Jain M. Wheelock C.E. Nilsson R. Metabolite profiling and stable isotope tracing in sorted subpopulations of mammalian cells.Anal. Chem. 2016; 88: 2707-2713Crossref PubMed Scopus (24) Google Scholar). To minimize cases where cells are in G1 phase during 13C-labeling but transition to S phase before sorting, we used a short (3 h) pulse in combination with the gating scheme described above (Figures 1B and 2A). We performed such isotope tracing experiments in normal human mammary epithelial cells (HMECs), H-rasV12-transformed HMECs (HMEC Ras) (Elenbaas et al., 2001Elenbaas B. Spirio L. Koerner F. Fleming M.D. Zimonjic D.B. Donaher J.L. Popescu N.C. Hahn W.C. Weinberg R.A. Human breast cancer cells generated by oncogenic transformation of primary mammary epithelial cells.Genes Dev. 2001; 15: 50-65Crossref PubMed Scopus (686) Google Scholar), and HeLa cells. Typically, several hundred putative metabolites exhibited 13C labeling (Table S1). For example, in HeLa cells, 921 high-quality LC-MS peaks were annotated with a putative metabolite identity (see STAR Methods), and of these, 546 (59%) had detectable 13C enrichment, indicating that they were synthesized by cells within the 3-h 13C pulse (Figures S2A and S2B). A total of 179 of these peaks (33%) reproducibly differed between G1 and SG2M cells by at least 5% in one or more mass isotopomer (MI) fractions (Figure 2B); a complete list is provided in Table S1. For example, metabolites of the pentose phosphate pathway (PPP), including 6-phosphogluconate and sedoheptulose-7-phosphate, were preferentially labeled in G1 (Figure 2C), suggesting increased activity of both the oxidative and non-oxidative PPP branches in this phase. These differences between G1 and SG2M were also seen when data were expressed as MI fractions, indicating that they are not due to changes in metabolite pool sizes (Figures S2C–S2F). These data may reflect PPP activity at the G1/S transition (Vizán et al., 2009Vizán P. Alcarraz-Vizán G. Díaz-Moralli S. Solovjeva O.N. Frederiks W.M. Cascante M. Modulation of pentose phosphate pathway during cell cycle progression in human colon adenocarcinoma cell line HT29.Int. J. Cancer. 2009; 124: 2789-2796Crossref PubMed Scopus (78) Google Scholar), since our gating scheme excludes early S-phase cells from the SG2M fraction. As a whole, these data indicate that a variety of metabolic differences exists between the SG2M and G1 phases in human cells. Beyond detecting synthesis of metabolites, MIs provided detailed information on the coordination of metabolic processes with the cell cycle. For example, UDP exhibited 13C5, 13C7, and 13C8 MIs typical of pyrimidine de novo synthesis in both G1 and SG2M cells (Figure 2D), indicating that de novo synthesis occurs throughout the cell cycle, while 13C deoxythymidine monophosphate (dTMP) was mainly formed in SG2M cells, as expected (Figure 2E). Moreover, most dTMP formed in SG2M was 13C-labeled on the methyl group within 3 h (indicated by a +1 shift of dTMP MIs compared to UDP), showing that both the dTMP pool and the upstream folate-bound one-carbon pool turns over rapidly in SG2M. Similarly, S-adenosylmethionine (SAM) was mostly 13C after 3 h, but most of this pool was 13C5, indicating that only the methionine group was labeled (Figure 2F), which shows that the SAM cycle turnover is much faster than de novo purine synthesis. In contrast to dTMP, formation of 13C5 SAM appears to be constant across the cell-cycle phases. Hence, the folate- and SAM-driven methylation systems are differently coordinated with the cell cycle. Among the hundreds of cell-cycle-associated metabolites, we noted a group consisting of arginine (Figure 3A) and several of its downstream metabolites (Figures 3D–3G), which acquired 13C label preferentially in the SG2M phase. Since arginine is essential for most cultured cells, increased 13C6 arginine in SG2M likely reflects increased uptake, which commonly occurs via the SLC7A1/2 transporter that also carries lysine (Schnorr et al., 2003Schnorr O. Suschek C.V. Kolb-Bachofen V. The importance of cationic amino acid transporter expression in human skin.J. Invest. Dermatol. 2003; 120: 1016-1022Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Consistent with this mechanism, we also observed higher 13C6 lysine in the SG2M phase (Figure 3B). Importantly, 13C6 arginine was low in non-transformed HMEC cells and did not increase in SG2M in this cell type (Figures 3A, S3A, and S3B). Although HMEC cells were cultured in a mammary epithelial cell medium (MCDB-170) with lower arginine concentration, transformed HMEC-Ras cells grown in MCDB-170 also showed high 13C6 arginine abundance, which again increased in SG2M (Figure 3A). This indicates that arginine uptake increases in the committed SG2M phase and is associated with transformation in the HMEC model. Besides being required for protein synthesis, arginine has several metabolic fates (Figure 3C), including nitric oxide (NO), creatine, and polyamines (Morris, 2007Morris Jr., S.M. Arginine metabolism: boundaries of our knowledge.J. Nutr. 2007; 137: 1602S-1609SCrossref PubMed Google Scholar). To identify reactions that might be important in the SG2M phase specifically for transformed cells, we compared 13C labeling of arginine metabolites between HeLa, HMEC, and HMEC-Ras cells. We did not observe 13C6 citrulline, indicating that NO synthase activity is minor (data not shown). 13C4 creatine was observed in HeLa cells (Figure 3D) but not in other cell types (data not shown), and was therefore not considered further. Polyamines were not measurable on our LC-MS system, but 13C4 acetyl-putrescine, a breakdown product of polyamines, was present and increased in the SG2M phase of all cell types (Figure 3E), consistent with synthesis and turnover of polyamines in SG2M in both transformed and non-transformed cells. In contrast, we observed increased 13C5 ornithine in SG2M in HeLa and HMEC Ras cells, but not in HMEC cells (Figure 3F), and these differences were also evident when viewed as MI fractions, indicating that they are not merely due to changes in metabolite pool size (Figure S3B). Because ornithine is the only known substrate for polyamine synthesis, which is required for cell proliferation (Landau et al., 2012Landau G. Ran A. Bercovich Z. Feldmesser E. Horn-Saban S. Korkotian E. Jacob-Hirsh J. Rechavi G. Ron D. Kahana C. Expression profiling and biochemical analysis suggest stress response as a potential mechanism inhibiting proliferation of polyamine-depleted cells.J. Biol. Chem. 2012; 287: 35825-35837Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), and since it was formed from arginine specifically in transformed cells, we chose to focus on this step of arginine metabolism. To determine more accurately the cell-cycle phase where arginine uptake and ornithine synthesis occurs, we analyzed cells harboring fluorophore-tagged endogenous Cyclin A2, whose expression allows separating cells into G1, S, and G2M phases (Cascales et al., 2017Cascales H.S. et al.Cyclin A2 localises in the cytoplasm at the S/G2 transition to activate Plk1.bioRxiv. 2017; https://doi.org/10.1101/191437Crossref Scopus (0) Google Scholar). Both arginine and ornithine were highest in G2M phase cells (Figure 3G), suggesting that uptake and catabolism of arginine occurs in this phase. These findings agree with previous reports that ornithine and putrescine are important for progression through the S and G2 phases (Anehus et al., 1984Anehus S. Pohjanpelto P. Baldetorp B. Långström E. Heby O. Polyamine starvation prolongs the S and G2 phases of polyamine-dependent (arginase-deficient) CHO cells.Mol. Cell. Biol. 1984; 4: 915-922Crossref PubMed Scopus (45) Google Scholar). In addition, arginase activity in cell lysates as well as cellular urea content was increased in SG2M phase cells obtained by DTB synchronization (Figures S3C–S3F). Given that ornithine is the only known source of polyamines in human cells (Coleman et al., 2004Coleman C.S. Hu G. Pegg A.E. Putrescine biosynthesis in mammalian tissues.Biochem. J. 2004; 379: 849-855Crossref PubMed Google Scholar), it was puzzling that ornithine did not acquire 13C label in non-transformed HMEC cells. We hypothesized that HMEC cells might synthesize ornithine through a slower reaction, which was not detected during the 3-h 13C pulse used in our sorting experiments, possibly from glutamine via ornithine aminotransferase (OAT) (Figure 3C). To address this possibility, we performed separate tracing experiments with 13C5-glutamine and 13C6-arginine tracers for 48 h in unsynchronized cultures of several cell types. In these conditions, both HMEC cells and non-transformed BJ fibroblasts exhibited ornithine (Figure 3H) as well as downstream acetyl-putrescine (Figure 3I) labeling from 13C5-glutamine, indicating that OAT is active in these cell types. At this time point, we also detected 13C ornithine and acetyl-putrescine from 13C6-arginine in all cell types tested (Figures 3H and 3I). This indicates that some arginase activity is present in all cell types, but the enzyme is most active in SG2M phase in HeLa and HMEC-Ras cells, as measured by 13C pulse labeling (Figure 3H). Importantly, we did not observe any ornithine or acetyl-putrescine labeling from 13C5-glutamine in the tumor-derived HeLa and MDA-MB-231 cell lines (Figures 3H, 3I, and S3G), indicating that there is no ornithine synthesis from glutamine via OAT in these cancer cells. Therefore, HeLa and MDA-MB-231 should be dependent on arginase in the SG2M phase. To identify specific enzymes or transporters that might be responsible for the observed metabolic events, we integrated our isotope tracing data with data on cell-cycle gene expression profiles (Kagawa et al., 2013Kagawa Y. Matsumoto S. Kamioka Y. Mimori K. Naito Y. Ishii T. Okuzaki D. Nishida N. Maeda S. Naito A. et al.Cell cycle-dependent Rho GTPase activity dynamically regulates cancer cell motility and invasion in vivo.PLoS One. 2013; 8: e83629Crossref PubMed Scopus (54) Google Scholar, Grant et al., 2013Grant G.D. Brooks 3rd, L. Zhang X. Mahoney J.M. Martyanov V. Wood T.A. Sherlock G. Cheng C. Whitfield M.L. Identification of cell cycle-regulated genes periodically expressed in U2OS cells and their regulation by FOXM1 and E2F transcription factors.Mol. Biol. Cell. 2013; 24: 3634-3650Crossref PubMed Scopus (126) Google Scholar, Sadasivam et al., 2012Sadasivam S. Duan S. DeCaprio J.A. 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This analysis revealed 202 enzyme-metabolite pairs where the enzyme exhibited cyclic expression and/or RNAi phenotypes, and the related metabolite differed by cell-cycle phase in our tracing experiments (Table S2), suggesting cell-cycle-regulated metabolic reactions. In particular, we found that both the arginine/lysine transporter SLC7A2 and the mitochondrial ARG2 showed RNAi cell-cycle phenotypes, suggesting that they may underlie arginine metabolism in SG2M. Also, ARG2 was clearly present in the cell lines studied (Figure 4A), and ARG2 protein as well as enzymatic activity was increased by oncogene transformation in both HMEC cells and fibroblasts (Figures 4A and 4B). In contrast, ARG1 is known to be expressed mainly in liver (Uhlen et al., 2010Uhlen M. Oksvold P. Fagerberg L. Lundberg E. Jonasson K. Forsberg M. Zwahlen M. Kampf C. Wester K. Hober S. et al.Towards a knowledge-based Human Protein Atlas.Nat. Biotechnol. 2010; 28: 1248-1250Crossref PubMed Scopus (1697) Google Scholar). We therefore focused on ARG2 as a likely candidate for the high arginase activity in SG2M. Since ornithine synthesis from arginine was consistently found in the committed SG2M phase in transformed cells, we reasoned that suppressing ARG2 might prevent cancer cell proliferation. Transient small interfering RNA (siRNA) knockdown of ARG2 (siARG2) reduced ARG2 protein by >95% in HeLa, MDA-MB-231, and MDA-MB-468 cells at 70 h (Figures S4A–S4C), and also reduced 13C5 ornithine, indicating that the ARG2 reaction was suppressed (Figure 4C). At 70 h, after ∼1 day of ARG2 suppression, cell number was consistently reduced by up to 50%, indicating that the ARG2 protein is required for proliferation in multiple transformed cell types (Figures 4D–4G). Since the duration of ARG2 suppression was roughly equal to the doubling time of these cells, a 50% reduction in cell number corresponds to nearly complete growth suppression. Moreover, the decrease in cell number was correlated with the degree of ARG2 knockdown (Figures 4D–4G and S4A–S4C). Interestingly, siARG2 cell cultures had fewer G1/G0 cells, and approximately 30% more cells at the G2M stage (Figures 4H and S4D), consistent with ornithine synthesis occurring mainly in G2 (Figure 3F). Moreover, an analysis of the transcriptional response to ARG2 knockdown based on the Connectivity Map dataset (Subramanian et al., 2017Subramanian A. Narayan R. Corsello S.M. Peck D.D. Natoli T.E. Lu X. Gould J. Davis J.F. Tubelli A.A. Asiedu J.K. et al.A next generation connectivity map: L1000 platform and the first 1,000,000 profiles.Cell. 2017; 171: 1437-1452.e17Abstract Full Text Full Text PDF PubMed Scopus (1272) Google Scholar) revealed Cyclin B2-dependent mechanisms as the top differentially expressed gene set (Figure 4I). These data suggest that ARG2 inhibition does not render cells quiescent, a source of resistance to many antiproliferative drugs (Jedema et al., 2003Jedema I. Barge R.M. Nijmeijer B.A. Willemze R. Falkenburg J.H. Recruitment of leukemic cells from G0 phase of the cell cycle by interferons results in conversion of resistance to daunorubicin.Leukemia. 2003; 17: 2049-2051Crossref PubMed Scopus (14) Google Scholar), but rather interferes with G2M phase progression. In contrast, arginine deprivation arrested cells in G1/G0 (Figure 4J), consistent with reports that arginine is sensed by mTOR-dependent mechanisms that arrest cells in this phase (Wang et al., 2015Wang S. Tsun Z.Y. Wolfson R.L. Shen K. Wyant G.A. Plovanich M.E. Yuan E.D. Jones T.D. Chantranupong L. Comb W. et al.Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1.Science. 2015; 347: 188-194Crossref PubMed Scopus (540) Google Scholar). Hence, ARG2 inhibition causes a phenotype distinct from arginine deprivation. To investigate whether cancer cells can compensate for loss of ARG2 by activating the OAT reaction, we next performed tracing with U-13C-glutamine in siARG2 cells. Neither ornithine nor acetyl-putrescine acquired label from 13C5-glutamine in ARG2 knockdown HeLa cells even at 70 h (Figure 4K), indicating that these cells are unable to compensate for loss of ARG2 activity using OAT. Moreover, siARG2 cells were depleted of 13C2 (acetyl-labeled) acetyl-putrescine (Figure 4L), suggesting defective polyamine synthesis downstream of ARG2. These results suggest that ornithine synthesis by ARG2 in the committed phase of the cell cycle might be a vulnerability of cancer cells (Figure 4M). To assess the role of ARG2 in human cancer, we analyzed its expression pattern in over 2,000 human breast tumors from multiple patient studies (see STAR Methods). We found ARG2 to be highly expressed (Figures 4N and S4E) and associated with poor patient survival (Figure S4F) in tumors with low estrogen receptor α (ER-α), but not in breast tumors overall. Hence, ARG2 is likely not a typical proliferation-associated gene that is broadly overexpressed in a wide variety of cancers (Selfors et al., 2017Selfors L.M. Stover D.G. Harris I.S. Brugge J.S. Coloff J.L. Identification of cancer genes that are independent of dominant proliferation and lineage programs.Proc. Natl. Acad. Sci. USA. 2017; 114: E11276-E11284Crossref PubMed Scopus (13) Google Scholar) but may be important in particular cell types. Accordingly, a recent study showed that, in clear-cell renal carcinomas, ARG2 expression is lower than in normal kidney, and in this context overexpression of ARG2 decreased cancer cell proliferation (Ochocki et al., 2018Ochocki J.D. Khare S. Hess M. Ackerman D. Qiu B. Daisak J.I. Worth A.J. Lin N. Lee P. Xie H. et al.Arginase 2 suppresses renal carcinoma progression via biosynthetic cofactor pyridoxal phosphate depletion and increased polyamine toxicity.Cell Metab. 2018; 27: 1263-1280.e6Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Hence, it is instrumental to identify specific cancer types that depend on ARG2 for growth. Toward this goal, we stratified breast tumors into normal-like, basal-like, Her2-positive, Luminal A and Luminal B subtypes using the PAM50 method (Parker et al., 2009Parker J.S. Mullins M. Cheang M.C. Leung S. Voduc D. Vickery T. Davies S. Fauron C. He X. Hu Z. et al.Supervised risk predictor of breast cancer based on intrinsic subtypes.J. Clin. Oncol. 2009; 27: 1160-1167Crossref PubMed Scopus (2955) Google Scholar). We observed high ARG2 expression specifically in basal-like tumors, which are considered aggressive and difficult to treat with existing drugs (Rakha et al., 2008Rakha E.A. Reis-Filho J.S. Ellis I.O. Basal-like breast cancer: a critical review.J. Clin. Oncol. 2008; 26: 2568-2581Crossref PubMed Scopus (697) Google Scholar), and occasionally in Her2-positive tumors (Figures 4O and S4G). This was likely not due to infiltration of fibroblasts or macrophages, since we found no correlation between ARG2 expression and expression of markers for these cell types in the tumor samples analyzed (Table S3). In a meta-analysis (Figure 4P), high ARG2 expression was also correlated with poor survival of patients specifically within basal-like breast cancer. Interestingly, the breast cancer cell lines for which we observed decreased proliferation upon ARG2 knockdown (Figures 4F and 4G) were derived from basal-like tumors as well. These results suggest that ARG2 should be considered a potential target for specific tumor types. In this paper, we have presented a large-scale investigation of metabolic events in the “committed” SG2M phase of the cell cycle, using a combination of cell sorting with mass spectrometry-based isotope tracing. A number of metabolites and enzymes were associated with cell-cycle phase and should be of great interest for future studies. In particular, we find that the mitochondrial ARG2 is active in the SG2M phase of cancer cells, but not in normal epithelial cells. Importantly, ARG2 appears to be the only source of ornithine in transformed cells, while normal cells can obtain ornithine via the OAT enzyme. This suggests that targeting ARG2 may disrupt polyamine synthesis specifically in transformed cells, which would be a major improvement on current strategies for targeting polyamine synthesis. Our results indicate that ARG2 suppression indeed reduces cell growth, without compensatory induction of OAT. However, it is important to note that ARG2 is expressed only in specific tumor types, including basal-like breast tumors, and additional studies are needed to investigate how specific tumor or cell types would respond to ARG2 inhibition." @default.
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• W2913854500 title "Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG2M Phase" @default.
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