RESEARCH METABOLISM identified 15N-isotopologs of proline, aspartate, branched-chain amino acids (BCAAs), and gluta- mate, which have no previous biosynthetic con- nection to the amide nitrogen on glutamine (Fig. Metabolic recycling of ammonia via 1C and fig. S3B). The labeled nitrogen was liber- ated as ammonia before production of these glutamate dehydrogenase supports metabolites, which suggests that an ammonia- recycling pathway may synthesize the other glu- breast cancer biomass tamine derivatives detected. To test whether ammonia released during glutaminolysis was necessary for production of Jessica B. Spinelli,1,2 Haejin Yoon,1 Alison E. Ringel,1 Sarah Jeanfavre,2 these unanticipated amide-nitrogen glutamine Clary B. Clish,2 Marcia C. Haigis1* derivatives, we treated cells with the glutamin- ase inhibitor bis-2-(5-phenylacetamido-1,3,4- Ammonia is a ubiquitous by-product of cellular metabolism; however, the biological thiadiazol-2-yl)ethyl sulfide (BPTES), which at consequences of ammonia production are not fully understood, especially in cancer. 1 mM is not cytotoxic or cytostatic in T47D and We found that ammonia is not merely a toxic waste product but is recycled into central MCF7 human breast cancer cell lines (16)(fig.S4, amino acid metabolism to maximize nitrogen utilization. In our experiments, human breast A to C). BPTES treatment significantly decreased cancer cells primarily assimilated ammonia through reductive amination catalyzed by 15N-isotopologs of glutamate, proline, and as- glutamate dehydrogenase (GDH); secondary reactions enabled other amino acids, such as partate, whereas metabolites involved in direct proline and aspartate, to directly acquire this nitrogen. Metabolic recycling of ammonia glutamine metabolism, such as nucleotides and accelerated proliferation of breast cancer. In mice, ammonia accumulated in the tumor Downloaded from asparagine, remained labeled (Fig. 1D and fig. microenvironment and was used directly to generate amino acids through GDH activity. S4D). Addition of ammonia to BPTES-treated These data show that ammonia is not only a secreted waste product but also a fundamental cells restored metabolites depleted by glutamin- nitrogen source that can support tumor biomass. ase inhibition, demonstrating the specific contri- bution of ammonia (fig. S4E). This is consistent ncreased nutrient consumption can supply assimilation: (i) carbamoyl phosphate synthe- with findings that ammonia partially rescues pro- carbon, nitrogen, oxygen, and sulfur to ac- tase I (CPS1), the adenosine triphosphate (ATP)– liferative defects in glutamine-deprived breast http://science.sciencemag.org/ commodate the extensive bioenergetic, bio- dependent, rate-limiting step of the urea cycle; cancer cells (17). I synthetic, and prosurvival requirements of (ii) glutamate dehydrogenase (GDH), a NADPH We examined the potential mechanisms under- rapidly proliferating cells (1–3). As a conse- (reduced nicotinamide adenine dinucleotide lying assimilation of ammonia liberated during quence, such cells generate an excess of metabolic phosphate)–dependent enzyme that catalyzes glutaminolysis. Because [15N]amide-glutamine waste products, which are cleared in mammals the reductive amination of a-ketoglutarate; and did not elicit any isotopes of four urea cycle in- through the excretory system. However, in the tu- (iii) glutamine synthetase (GS), which catalyzes termediates (ornithine, citrulline, argininosuc- mor microenvironment, metabolic waste products the ATP-dependent amination of glutamate to cinate, and arginine), we ruled out the activity such as lactate and ammonia accumulate (4, 5). generate glutamine (12, 13) (fig. S1A). Analysis of CPS1 as a mechanism for ammonia assimi- Although lactate is well studied in cancer, little is of transcriptomic data from The Cancer Genome lation (fig. S2). Instead, our data indicated that known about the mechanisms by which cancer Atlas for the ammonia-assimilating enzymes in GDH was the primary point of ammonia as- cells manage increased amounts of ammonia healthy and cancerous tissues revealed that ex- similation because glutamate is upstream of on June 17, 2019 (NH3) generated by glutamine and asparagine pression of GS and GDH mRNA is significantly proline, aspartate, glutamine, and BCAA syn- catabolism, de novo cysteine synthesis through the increased across many cancer subtypes, whereas thesis. However, the Michaelis-Menten constant transsulfuration pathway, and salvage nucleotide CPS1 mRNA is increased only in the colon (Fig. 1B). (Km) of GDH for ammonia is high (approximate- metabolism (6). Ammonia has been considered a Amonghealthytissues,GSandGDHareubiqui- ly 9 mM) and GDH reportedly favors oxidative toxic by-product that must be exported from cells tously expressed and CPS1 is expressed only in the deamination over reductive amination in can- and is subsequently cleared through urea cycle liver (fig. S1B). Breast cancers display increased cer cells (18–21). By contrast, GDH-catalyzed re- activity in the liver (7–9). expression of both GS and GDH. Specifically, es- ductive amination is prevalent in the liver, where Glutamine has been called a “nitrogen reser- trogen receptor–positive (ER+) breast cancers have there is a sufficient concentration of ammonia voir” for cancer cells because of its anabolic role in increased expression of GS and GDH relative to to enable catalysis in this direction (22). We nucleotide synthesis (6, 10). However, the role of that in other subtypes (14). Therefore, we used wondered whether increased concentrations glutamine as a nitrogen reservoir is contradicted ER+ breast cancer as a representative model to of ammonia in the tumor microenvironment in catabolic glutamine metabolism, because nitro- probe for ammonia assimilation. might also permit GDH-catalyzed reductive gen is liberated as the by-product ammonia (11). To investigate the fate of glutamine-derived amination. The fate of ammonia in the metabolism of pro- ammonia, we performed a metabolic tracing anal- To determine whether GDH assimilates ammo- liferating cells and tumors remains unclear. We ysis with hydrophilic interaction liquid chroma- nia generated by glutamine catabolism, we used hypothesized that ammonia might be reassimi- tography tandem mass spectrometry (HILIC-MS) short hairpin RNA (shRNA) to deplete cells of lated into central metabolism to maximize the and assessed the fate of [15N]amide-glutamine, GDH, cultured them with [15N]amide-glutamine, 15 efficiency of nitrogen utilization. In this study, we which liberates [ N]NH3 through glutaminase and then subjected them to nitrogen metabolome sought to clarify the role of ammonia as either a activity (15). To identify the metabolic derivatives scanning (fig. S4F). MCF7 and T47D cell lines toxic waste product or a biosynthetic metabolite of [15N]amide-glutamine in an unbiased manner, express both GDH1 and GDH2 isoforms, and (Fig. 1A). we developed a method to screen the nitrogen shRNAs targeted both isoforms (fig. S4G). The Mammals have three enzymes that can over- metabolome, which contained 211 15N-isotopologs abundance of 15N-isotopologs of glutamate and come the thermodynamic hurdles of ammonia (table S1). The majority of the nitrogen meta- downstream metabolites (proline, aspartate) bolome did not acquire 15N labeling; of 211 15N- was significantly decreased in cells depleted of isotopologs, only 33 metabolites were labeled (fig. GDH (Fig. 1E). Urea cycle intermediates (citrul- 1 Department of Cell Biology, Harvard Medical School, Boston, S2). Consistent with previous studies, [15N]amide- line, argininosuccinate) remained unlabeled in MA 02115, USA. 2Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. glutamine was incorporated into asparagine and cells lacking GDH, underscoring the lack of *Corresponding author. Email: [email protected] nucleotides (10) (Fig. 1C and fig. S3A). We also CPS1-mediated ammonia assimilation in breast Spinelli et al., Science 358, 941–946 (2017) 17 November 2017 1of6 RESEARCH | REPORT cancer cells (Fig. 1E). Reexpression of shRNA- from [15N]amide-glutamine (23) to measure the (fig. S6, A to C). Because ~2% of the glutamate pool 15 insensitive GDH1 rescued labeling onto glutamate amount of [ N]NH3 generated after 8 hours of acquired this label in a glutaminase-dependent and downstream metabolites (fig. S5). treatment with [15N]amide-glutamine. In MCF7 manner (fig. S3B), we hypothesized that ammo- Next, we used a liquid chromatography–mass and T47D cells, we found that only 3.5% of the nia recycling from glutaminolysis is highly effi- 15 spectrometry method for the detection of [ N]NH3 total ammonia pool derived from glutaminolysis cient. We quantified the efficiency of ammonia Assimilation Waste Fold-Change Expression Compared to Healthy Tissue ABCDEFGHIA JK LM GS 1.50 1.16 1.12 1.16 1.15 1.06 -1.15 1.10 1.27 1.11 ? GDH 1.34 1.32 1.25 1.22 1.03 -2.17 NH 3 CPS1 1.36 1.05 OO OO Sample Size 594 102 60 69 331 471 360 38 173 542 18 138 58 - (Patients) H N O -O O- 2 + NH + 3 NH3 Glutamine Significantly Not Significantly Significantly Glutamate Decreased Altered Increased -O O O O O O HN T47D MCF7 Downloaded from P O O O - - N O O H2N N O OH H 2.0 (-) BPTES O- 3.0 OH N-Carbamoyl Aspartate **** (+) BPTES HO NH Proline (M+1) **** 1.5 UMP (M+1) 2.0 **** **** O O 1.0 O P **** **** - 1.0 O H2N O O O- O- O 0.5 H N Isotopologue 2 - - O carbamoyl phosphate Abundance (%) O http://science.sciencemag.org/ N O- O NH + 0.0 3 + 0.0 Asparagine (M+1) O NH3 NH3 Aspartate (M+1) Glu Pro Asp Glu Pro Asp OO O O - -O O- α H2N O -ketoglutarate + GLS + NH NH3 T47D Aspartate 3 Glutamate (M+1) Glutamine O 1.5 shControl - **** HO O O- **** shGDH #1 P O NH2 PRPP O NH + **** shGDH #2 FGAR O 3 **** Leucine (M+1) OH 1.0 **** Gln HO **** - N HO O H H2N - N O O OH 0.5 P O O N H N O O P on June 17, 2019 O Isotopologue NH N O OH N Abundance (%) HO HO NDNDND NDND ND OH 0.0 AMP (M+2) Unexpected Derivatives Glu Pro Asp Cit Asa 15NH 3 Ammonia Recycling Direct Pathways OO O O O O 15 O- - Nucleotides H2 N HO O -O O- GLS 15 GDH + + NH3 NH3 O Glutamine Glutamate α-Ketoglutarate Asparagine Proline Aspartate BCAAs Fig.