Mitochondrial One-Carbon Metabolism Maintains Redox Balance During Hypoxia Inmaculada Martínez-Reyes and Navdeep S

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IN THE SPOTLIGHT Mitochondrial One-Carbon Metabolism Maintains Redox Balance during Hypoxia Inmaculada Martínez-Reyes and Navdeep S. Chandel Summary: Mitochondria generate high levels of reactive oxygen species (ROS) to activate protumorigenic signaling pathways. In parallel, the mitochondria must also increase their antioxidant capacity to lower ROS levels and prevent cancer cell death. In this issue of Cancer Discovery, Ye and colleagues demonstrate that serine catabolism through one-carbon metabolism within the mitochondrial matrix is necessary to maintain this redox balance. Cancer Discov; 4(12); 1371–3. ©2014 AACR.

See related article by Ye et al., p. 1406 (2).

Cancer cell proliferation requires a robust increase in THF). Cytosolic and mitochondrial methylenetetrahydrofolate

cellular metabolism to support the massive anabolic require- dehydrogenase (MTHFD1 and 2, respectively) use 5,10-CH2 - ments for the generation of two daughter cells. Cancer cells THF and NADP+ as substrates to produce 5,10-methenyl- engage in multiple glucose- and mitochondrial-dependent tetrahydrofolate (5,10-CH=THF) and NADPH ( Figure 1 ). anabolic pathways to generate the precursors required for Subsequently, 5,10-CH=THF is converted into 10-formyl-THF, lipid, nucleotide, and protein synthesis as well as to produce which is used for purine synthesis. Thus, serine catabolism NADPH, which provides the reducing equivalents for bio- through one-carbon metabolism supports cancer cell prolif- synthesis. Oncogenic signaling and tumor-suppressor loss eration ( 3 ). Recently, several studies have highlighted the role activate these anabolic pathways to support tumor growth. of serine in tumorigenesis. For example, the initial cytosolic Indeed, as a consequence of mitochondrial metabolism, the enzyme in the de novo serine synthesis pathway, phosphoglycer- reactive oxygen species (ROS) generated by the mitochondrial ate dehydrogenase (PHGDH), is upregulated in breast cancer electron transport chain (ETC) are essential for cancer cell and melanoma ( 4, 5 ). Moreover, many tumor cells are highly proliferation, tumorigenesis, and metastasis ( 1 ). When rap- dependent on the uptake of exogenous serine, suggesting that idly proliferating tumor cells outgrow their available blood the de novo serine synthesis by itself is not sufﬁ cient to meet the supply, regions within a solid tumor become hypoxic (i.e., requirements for cell proliferation ( 6 ). have low oxygen levels). Hypoxia also increases the produc- Given that one-carbon THF units are required for nucle- tion of mitochondrial ROS to activate the HIF family of otide synthesis, cancer cells beneﬁ t from enhanced serine- transcription factors and induce the expression of HIF target dependent one-carbon metabolism. Notably, serine is genes, including those involved in metabolism and angiogen- primarily catabolized through the mitochondrial one-carbon esis. Importantly, cancer cells need to maintain a steady-state metabolism pathway. If carbon units of THF are needed solely level of ROS, a redox balance, which allows for cell prolifera- for nucleotide synthesis in the cytosol, why do cells engage in tion and HIF activation without allowing ROS to accumulate the mitochondrial one-carbon metabolism pathway? A recent to levels that would incur cell death or senescence. Thus, elegant study that uses a new method for tracing NADPH mitochondrial ROS levels are tightly regulated in cancer compartmentalization revealed that serine functions in the cells. Ye and colleagues demonstrate that serine catabolism mitochondrial one-carbon metabolism pathway to produce through one-carbon metabolism maintains this mitochon- NADPH (7 ). An independent study also demonstrated that drial redox balance during hypoxia ( 2 ). serine and glycine catabolism in the mitochondria gener- During one-carbon metabolism, serine is converted to ates NADPH (8 ). However, whether this source of NADPH glycine in the cytosol and mitochondrial matrix by serine is important for cancer cell proliferation and tumor growth hydroxymethyl transferase 1 and 2 (SHMT1 and 2), respec- remained unknown. tively. This reaction involves the covalent linkage of tetrahy- In this issue of Cancer Discovery , Ye and colleagues ( 2 ) drofolate (THF), derived from folic acid, to a methylene group not only describe the importance of the mitochondrial one-

(CH2 ) to form 5,10-methylene-tetrahydrofolate (5,10-CH2 - carbon metabolism pathway but also provide mechanistic insight into the role of serine in NADPH production for mitochondrial redox homeostasis during hypoxia and tumor Department of Medicine, Northwestern University Feinberg School of growth. NADPH plays a critical role in maintaining the cel- Medicine, Chicago, Illinois. lular antioxidant capacity by regenerating the reduced pools Corresponding Author: Navdeep S. Chandel, Department of Medicine, of glutathione (GSH) and thioredoxin (TRX), ROS scavengers Northwestern University Feinberg School of Medicine, Chicago, IL 60611. that remove excess hydrogen peroxide (H2 O2 ). Ye and col- Phone: 312-503-2549; Fax: 312-503-0411; E-mail: [email protected] leagues observed a drastic increase in the amount of mito- doi: 10.1158/2159-8290.CD-14-1228 chondrial ROS produced in SHMT2-knockdown cells under ©2014 American Association for Cancer Research. hypoxia compared with normoxia. Moreover, these cells had

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Glucose PHGDH Purines 3-P-Glycerate SSerinee GlycineGlyc SHMT1 MTHFD1 10-formyl1 yl THF 5,10-CH2-THFF -THF FFormate One-carbon metabolism Pyruvateuva TCA Cycle SerineSerrineri GlyGlycine –. –. O2 VDACs O2 NADH NAD+ SHMT2 MTHFD2 10-formyl SOD1 FormateFForm ETC THFHF 5,10-CH2-THFF -THF O2 + H2O2 NADP NADPH Redox balance

Redox GSH TRX H O H O signaling 2 2 2 SOD2 O –. MYC HIF1 2 O2 ETC

− Figure 1. Serine catabolism maintains redox balance during hypoxia. Hypoxia triggers the production of superoxide (O2 •) in the mitochondria by ETC. − O 2 • is transported to the cytosol through voltage-dependent anion channels (VDAC) and is converted into hydrogen peroxide (H2 O 2 ) by SOD1. H 2O 2 acts − as a signaling molecule to promote cancer cell proliferation and HIF activation. O2 • can also be converted to H2 O 2 in the mitochondrial matrix by SOD2 and further detoxiﬁ ed to water (H2 O) by glutathione peroxidases (GPX) and peroxiredoxins (PRX) to maintain the redox balance. The scavenging ability of GPX and PRX is dependent on glutathione (GSH) and thioredoxin (TRX). NADPH is used to regenerate the reduced glutathione (GSH) and thioredoxin (TRX) pools. Serine catabolism within the mitochondrial matrix can replenish NADPH. The de novo serine synthesis is initiated in the cytosol by phosphoglycerate dehydrogenase (PHGDH) using the glycolytic intermediate 3-P-glycerate. Serine is catabolized by the cytosolic (SHMT1) and mitochondrial + = matrix (SHMT2) enzymes to produce glycine and 5,10-CH2 -THF. The MTHFD enzymes further convert 5,10-CH2 -THF and NADP into 5,10-CH THF and NADPH. Subsequently, 5,10-CH=THF is converted into 10-formyl-THF, which is used for purine synthesis. The transcription factors HIF1 and MYC coop-

erate to upregulate SHMT2 in cancer cells during hypoxia to prevent uncontrolled levels of H 2O 2 in the mitochondrial matrix that would be detrimental for cancer cells.

lower NADPH/NADP+ and glutathione/glutathione disulfi de cation. Because SHMT2 is a reported MYC target gene, Ye and (GSH/GSSG) ratios during hypoxia, resulting in increased cell colleagues further investigated whether MYC was also driving death. Importantly, this effect was rescued when the cells were SHMT2 upregulation. Indeed, they confi rmed that the induc- treated with the antioxidant N-acetylcysteine (NAC), implicat- tion of SHMT2 under hypoxia was dependent on MYC-driven ing elevated ROS in the increased cell death of SHMT2-knock- transcriptional amplifi cation, indicating that both HIF and down cells. Furthermore, cancer cells with decreased SHMT2 MYC collaborate to induce SHMT2 expression (Figure 1). also display impaired tumor growth. Thus, mitochondrial This fi nding was quite unexpected as previous results in serine catabolism is necessary for NADPH production, ROS clear cell renal cell carcinoma have demonstrated that HIF1α detoxifi cation, and cancer cell survival. Importantly, SHMT2 antagonizes c-MYC function ( 9 ). Further studies are neces- but not SHMT1 is overexpressed in a variety of human can- sary to elucidate the circumstances for which HIF1 and MYC cers, and breast cancer patients with low SHMT2 expression cooperate or antagonize to regulate gene expression. survive better compared to those with high SHTM2 expres- The degree of hypoxia positively correlates with metastasis; sion. In addition, there is a positive correlation between mito- thus, it will be of interest to determine whether enzymes in chondrial SHMT2 (serine catabolism) and cytosolic PHGDH the one-carbon metabolism pathway predict metastatic pro- (serine biosynthesis). This correlation is not observed between gression. It will also be important to assess whether targeting the two cytosolic proteins, PHGDH and SHMT1. This sug- these metabolic enzymes diminishes both primary tumor gests that cancer cells coordinate the de novo synthesis of growth and metastasis. Moreover, given that MYC expres- serine in the cytosol and the catabolism of serine in the mito- sion is deregulated in numerous tumor types, the fi nding chondrial matrix to produce NADPH for detoxifi cation of that SHMT2 induction occurs only in MYC -amplifi ed cancer ROS needed to sustain tumor growth (Figure 1). cells during hypoxia makes SHMT2 an attractive therapeutic In addition, Ye and colleagues observed that the expression target. Furthermore, p53-null tumors are highly sensitive to of SHMT2 positively correlated with the expression of HIF- exogenous serine depletion ( 6 ), suggesting that mitochon- regulated enzymes under hypoxia and confi rmed hypoxia- drial one-carbon metabolism may prove to be an effi cacious induced upregulation of SHMT2 in a HIF1-dependent cancer therapy in MYC-overexpressing and p53-null tumors. manner in cancer cells ( 2 ). Interestingly, hypoxic induction of Interestingly, anti-folate agents that disrupt one-carbon SHMT2 was observed only in cancer cells with MYC amplifi - metabolism (folic acid is the precursor for the one-carbon

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unit donor THF) have been used clinically for decades and Disclosure of Potential Confl icts of Interest thus there is precedence for targeting this pathway. No potential confl icts of interest were disclosed. A key concern with inhibiting mitochondrial one-carbon metabolism is whether cancer cells exhibit metabolic plas- Grant Support ticity to compensate for the loss of NADPH production. It N.S. Chandel is supported by the NIH (grant R01 CA12306708). is important to note that within the mitochondrial matrix I. Martínez-Reyes is supported by the Ramon Areces Foundation of NADPH can also be generated by isocitrate dehydrogenase Spain. 2 (IDH2) and malic enzyme 3 (ME3). Another important consideration is whether normal cells require mitochon- Published online December 4, 2014. drial one-carbon metabolism for maintaining redox balance. Although Shmt2 -knockout mice have yet to be generated, Mthfd2-knockout mice are embryonic lethal. Given the REFERENCES hypoxic microenvironment of a developing embryo, it is not 1. Schieber M , Chandel NS . ROS function in redox signaling and oxida- surprising that mice lacking a key enzyme within the mito- tive stress . Curr Biol 2014 ; 24 : R453 – 62 . chondrial one-carbon metabolism pathway are embryonic 2. Ye J , Fan J , Venneti S , Wan Y-W , Pawel BR , Zhang J , et al. Serine catabolism regulates mitochondrial redox control during hypoxia . lethal. However, normal adult tissues might not require these Cancer Discov 2014;4:1406–17 . mitochondrial one-carbon metabolism enzymes, as mito- 3. Labuschagne CF , van den Broek NJ , Mackay GM , Vousden KH , Mad- chondrial oxidative stress is low and nutrients such as glucose docks OD . Serine, but not glycine, supports one-carbon metabolism and oxygen are not limiting. Indeed, MTHFD2 was absent and proliferation of cancer cells. Cell Rep 2014 ; 7 : 1248 – 58 . in most adult tissues, in stark contrast with the developing 4. Locasale JW , Grassian AR , Melman T , Lyssiotis CA , Mattaini KR , Bass embryo ( 10 ). Meta-analysis data showed that MTHFD2 is AJ , et al. Phosphoglycerate dehydrogenase diverts glycolytic fl ux and the most consistently overexpressed metabolic enzyme across contributes to oncogenesis. Nat Genet 2011 ; 43 : 869 – 74 . 5. Possemato R , Marks KM , Shaul YD , Pacold ME , Kim D , Birsoy K, multiple tumor types and is correlated with poor survival in et al. Functional genomics reveal that the serine synthesis pathway is breast cancer (10 ). Thus, targeting mitochondrial one-carbon essential in breast cancer. Nature 2011 ; 476 : 346 – 50 . metabolism for cancer treatment is a potential therapeutic 6. Maddocks OD , Berkers CR , Mason SM , Zheng L , Blyth K , Gottlieb E , approach. However, a major hurdle for inhibitors targeting et al. Serine starvation induces stress and p53-dependent metabolic this pathway is diffusibility through the lipid membranes, remodelling in cancer cells. Nature 2013 ; 493 : 542 – 6 . including the cell membrane, outer mitochondrial mem- 7. Lewis CA , Parker SJ , Fiske BP , McCloskey D , Gui DY , Green CR, et al. Tracing compartmentalized NADPH metabolism in the cytosol and branes, and inner mitochondrial membranes. To overcome mitochondria of mammalian cells . Molecular Cell 2014 ; 55 : 253 – 63 . this obstacle, a lipophilic cation can be attached to small 8. Fan J , Ye J , Kamphorst JJ , Shlomi T , Thompson CB , Rabinowitz JD . molecules to increase their accumulation in the mitochon- Quantitative fl ux analysis reveals folate-dependent NADPH produc- drial matrix 100- to 500-fold greater in concentration than tion. Nature 2014 ; 510 : 298 – 302 . outside the cell due to the large negative membrane potential 9. Keith B , Johnson RS , Simon MC . HIF1alpha and HIF2alpha: sibling generated by the ETC across the mitochondrial inner mem- rivalry in hypoxic tumour growth and progression . Nat Rev Cancer brane. Thus, in summary, Ye and colleagues ( 2 ) have unveiled 2012 ; 12 : 9 – 22 . 10. Nilsson R , Jain M , Madhusudhan N , Sheppard NG , Strittmatter L , mitochondrial one-carbon metabolism as a promising path- Kampf C , et al. Metabolic enzyme expression highlights a key role for way for targeted cancer therapy, especially in hypoxic tumors MTHFD2 and the mitochondrial folate pathway in cancer . Nat Com- where, to date, the therapeutic options are limited. mun 2014 ; 5 : 3128 .

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Inmaculada Martínez-Reyes and Navdeep S. Chandel

Cancer Discovery 2014;4:1371-1373.

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