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. 2019 Feb 12;26(7):1691-1700.e5.
doi: 10.1016/j.celrep.2019.01.059.

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG 2 M Phase

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Free PMC article

Mapping Metabolic Events in the Cancer Cell Cycle Reveals Arginine Catabolism in the Committed SG 2 M Phase

Irena Roci et al. Cell Rep. .
Free PMC article

Abstract

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.

Keywords: ARG2; OAT; arginase 2; basal-like breast cancer; cancer metabolism; isotope tracing; mass spectrometry; ornithine; polyamines.

Figures

Figure 1
Figure 1. A cell sorting approach to studying metabolism in the G1 and SG2M phases.
(A) Design of cell sorting and LC-MS experiments. (B) Gating strategy for isolating G1 and SG2M cells, illustrated by Hoechst staining vs. Geminin probe expression in HeLa cells. Blue and red highlighted regions indicate gates for 2n (G1) and 4n (SG2M), respectively. (C) Western blot of cell cycle phase markers Cdt1 (G1) and Cyclin A (SG2M) in sorted HeLa cell populations. (D–E) Relative abundance (LC-MS peak area) of deoxythymidine triphosphate (dTTP) and deoxyadenosine triphosphate (dATP) in sorted G1 and SG2M phase HeLa cells (D), and in unsynchronized, double thymidine block (DTB) and lovastatin (LOV) synchronized HeLa cells (E). (F–H) Relative abundance of ADP-ribose (F), ribose/ribulose-5-phosphate (G), and sedoheptulose-7-phosphate (H), in synchronized and sorted HeLa cells, normalized to the mean of LOV and G1 samples, respectively. See also Figure S1.
Figure 2
Figure 2. Pulse 13C labeling of sorted cells identifies cell cycle-regulated pathways.
(A) Left, experimental design of pulse labeling followed by cell sorting and LC-HRMS. Asterisk (*) denotes 13C isotopes. Right, cell cycle diagram indicating gating for 2n (G1) cells (blue arc), and 4n (SG2M) cells (red arc) based on Hoechststaining. Grey circle sections represent example cell cycle trajectories during a 3 hour incubation with 13C labeled medium, designed to minimize cross-contamination (see text). (B) Distribution of difference in mass isotopomer (MI) fraction between SG2M and G1 cells. Solid black line denotes mean of biological triplicates, gray denotes standard deviation. Highlighted metabolites, see text. (C) Selected MI peak areas for pentose phosphate pathway metabolites in G1 (blue) and SG2M (red) cells. Error bars denote standard deviation of biological triplicates. (D–E) Above, MI peak areas of uridine-diphosphate (UDP) (D) and dTMP (E) in G1 (blue) and SG2M (red) cells. Paired data from three independent sorting experiments are shown, with adjacent bars from the same experiment. Below, UDP and dTMP structures with origin of observed MIs indicated. dTMP derives from UDP, with one methyl group added from CH2-folate by thymidylate synthase (TYMS). (F) MI peak areas and structure of S-adenosyl methionine (SAM), as in (D,E). See also Figure S2 and Table S1.
Figure 3
Figure 3. Argnine metabolism and ornithine synthesis occurs preferentially in SG2M.
(A–B) LC-MS peak areas of 13C6 arginine (A) and 13C6 lysine (B). Paired data from three independent sorting experiments are shown, with adjacent bars from the same experiment. (C) Diagram of metabolic reactions downstream of arginine. Metabolites that were not observed are shown in gray. Black and white circles indicate 13C and 12C atoms, respectively, as observed in (A–F). (D-F) LC-MS peak areas of, 13C4 creatine (D), 13C6 acetyl-putrescine (E) and 13C5 ornithine (F), in G1 (blue) and SG2M (red) phase HeLa, HMEC and HMEC Ras cells. (G) Abundance of arginine (arg), lysine (lys), ornithine (orn) and acetyl-putrescine (aptrc) in S and G2M cells, relative to G1. (H-I) 13C5 ornithine (H) and 13C4 acetylputrescine (I) MI fractions from 13C6-arginine and 13C5-glutamine tracing experiments at 48 hours, in indicated cell lines. Biological triplicates are shown for each sample in (H–I); error bars represent standard deviation. See also Figure S3.
Figure 4
Figure 4. ARG2 is important for proliferation of transformed cells.
(A) Western blot of ARG2 protein (size 39 kDa) in lysates of indicated cell types. Beta tubulin (TUBB gene product, size 50kDa), was used as a loading control. (B) Arginase activity in lysates of indicated cell types as measured by absorbance. Two independent experiments are shown for each sample. (C) Relative peak areas of 13C6 arginine and 13C5 ornithine in ARG2 knockdown samples. Data is presented as ratio over siCtrl samples. Two independent experiments are shown for each sample. (D–G), Relative cell numbers at 70 hours in cultures of untreated cells (Ctrl), cells treated with scrambled siRNA (siCtrl) or with siRNAs against ARG2 (siA, siB, si2, si3), in HeLa (D), HeLa Fucci (E), MDA-MB-231 (F) and MDA-MB-468 cells (G), in all cases normalized to Ctrl. (H) Fractions of HeLa cells in the G1, S and G2M phases as determined by DNA analysis, in cultures treated with siCtrl or siB. (I) Gene set analysis of mRNAs responding to ARG2 knockdown, from the ConnectivityMap dataset. Left, enrichment score vs. false discovery rate (FDR) for 3,582 gene sets, top gene set indicated. Right, individual gene ranks for the top gene set. (J) Fraction of G1 population in HCT116 (left) and MCF7 (right) cells grown in control (grey bars) and arginine deprived (black bars) medium. Two independent experiments are shown for each sample. (K) 13C5 ornithine and 13C4 acetyl-putrescine MI fractions in Ctrl and siB treated HeLa cells, labeled from 13C6-arginine (arg) or 13C5-glutamine (gln) tracers, as indicated. (L) LC-MS chromatogram of 13C2 (acetyl-labeled) acetyl-putrescine in Ctrl and siB treated HeLa cells labeled from 13C5-glutamine. (M) Model of arginine metabolism based on the experimental results. (N) ARG2 expression in the Miller et al. (GSE3494) breast tumor dataset, stratified by above-median (high) or below-median (low) estrogen receptor α (ESR1) expression. Affymetrix probe set accession number is indicated; p-value indicates one-sided permutation test. (O) ARG2 expression in breast cancer subtypes based on PAM50 classification, as in (H). (P) Meta-analysis of ARG2 expression association with patient survival in basal breast cancers, from indicated data sets. Error bars in A–C and E denote standard deviation across three experiments. Biological triplicates are shown for each sample in (B-J) and (K); error bars represent standard deviation. See also Figures S4 and Table S2.

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