COMMENTARY COMMENTARY Arginine deprivation and autophagic cell death in cancer Peter Wojciech Szlosarek1 Barts Cancer Institute, Queen Mary University of London, Center for Molecular Oncology, John Vane Science Center, London EC1M 6BQ, United Kingdom

Our increasing awareness over the last de- in a cell type-dependent manner (13–15). In cade, that reprogramming of cellular ener- PNAS, Changou et al. (16) describe a novel getics is a key hallmark of tumorigenesis, has reactive oxygen species (ROS)-mediated reinvigorated the search for novel tractable mechanism involved in ADI-PEG20–induced metabolic targets in cancer cells (1). From cell death in arginine-dependent (auxotro- − innovative diagnostics based on the tracer phic) ASS1 prostate cancer cell lines, defined 18F-deoxyglucose in positron emission to- by mitochondrial damage, nuclear DNA mography (FDG-PET) to the development leakage, and chromatin autophagy (“self-eat- of next-generation antimetabolites built on ing”), termed chromatophagy. This atypical L-asparaginase, methotrexate, and 5-fluoro- form of autophagic cell death (type 2 cell uracil, there is excitement that a deeper un- death) contrasts sharply with their earlier derstanding of tumor metabolism will revo- work in prostate cancer cells, showing that lutionize patient care (2). Modulating the key early-onset autophagy is a protective mecha- substrates glucose and glutamine has great nism in arginine-auxotrophic tumor cells that appeal because these provide the essential otherwise undergo apoptosis via a caspase- Fig. 1. Biphasic autophagic response following arginine carbon source for many anabolic cellular pro- independent mechanism (15). The new data deprivation in ASS1-ve tumor cells: prosurvival phase cesses required during tumor growth (3). followed by excessive autophagy leading to cell death. A, by Changou et al. (16) highlight that there is autophagosome; AL, autolysosome; CAT, cationic amino However, it has become apparent that there still much to learn about the effects of argi- acid transporter; GA, giant autophagosome; L, lysosome; aremanyothermetabolitesfuelingtumori- nine in cancer cells, and how we should ex- M, mitochondrion; N, nucleus. ASS1-ve tumor cells are genesis. These metabolites range from the ploit arginine starvation and autophagy in sensitive to the arginine-depletor ADI-PEG20 and ex- rare “oncometabolite” 2-HG in isocitrate de- combination with other therapies. hibit caspase-dependent or -independent apoptosis associated with DNA leakage. hydrogenase mutant tumors to critical roles Several groups have studied autophagy for a wider array of amino acids, including induced by arginine depletors and it is clear serine, glycine, proline, and especially argi- that many cancer cell types use this homeo- promotes apoptosis and has been suggested as nine (4–7). Targeting arginine has been the static recycling mechanism to overcome the a potential strategy going forward into clinical focus of preclinical and promising clinical arginine supply problem. The process begins trial (15, 18, 19). Alternatively, cleavage of activity driven by the availability of novel by enclosing macromolecules and organelles Beclin-1 and Atg5 by tumor necrosis factor- arginine depletors and an appreciation that within double membrane-bound vesicles, related apoptosis-inducing ligand has been a range of hematological and solid cancers known as the autophagosomes, which are shown to enhance apoptosis in ADI-treated are defective in urea cycle enzymes, namely then fused with lysosomes before disassembly melanoma cell lines via caspase-8 (20). Spe- argininosuccinate synthetase 1 (ASS1) and by hydrolases. Work in simple organisms has cific pharmacologic inhibitors of the autoph- argininosuccinate lyase (8, 9). Arginine is a identified a family of autophagy-related genes agy machinery (e.g., Atg proteins) await highly versatile amino acid required for the (Atg) that are activated following nutrient further development and may be more effec- synthesis of proteins, nitric oxide, polyamines, deprivation stress secondary to loss of the tive in combination with arginine depletors. nucleotides, agmatine, creatine, proline, and inhibitory effect of mTOR on Atg13, leading Although autophagy has an undoubted pro- glutamate. In particular, deficiency of the tu- to autophagosome formation. Effectively, survival function in tumors exposed to vari- — mor suppressor ASS1 frequently caused by ADI-induced autophagy buys tumor cells ous metabolic stresses, studies have revealed — methylation of the gene promoter confers a time before additional mechanisms are paradoxically a role in the regulation of cell worse prognosis and sensitizes tumors to ar- activated, such as ASS1 reexpression (7, 17). death. This finding is best illustrated by genetic – ginine starvation with pegylated arginine dei- ADI-PEG20 sensitive tumor cell lines ex- studies involving deletion of the autophagic minase (ADI-PEG20) and human recombi- hibit various markers of autophagy, including modulators beclin1, atg5,andatg7, which pre- – nant arginase (10 13). Arginine withdrawal conversion of cytosolic LC3-1 protein to the dispose mice to spontaneous tumorigenesis, leads to increased protein turnover—via re- lipidated form LC3-II, up-regulation of Beclin duced synthesis and increased breakdown 1 (Atg6), Atg genes 5 and 7, appearance of [suppression of mammalian target of rapamy- autophagosomes, and degradation of the auto- Author contributions: P.W.S. wrote the paper. cin (mTOR) and proteosomal degradation, phagy substrate protein p62 (18). Moreover, The author declares no conflict of interest. respectively]—and triggers caspase-dependent blocking autophagy with the autophagoso- See companion article 10.1073/pnas.1404171111. and caspase-independent apoptotic cell death mal–lysosomal fusion inhibitor, chloroquine, 1Email: [email protected].

www.pnas.org/cgi/doi/10.1073/pnas.1416560111 PNAS Early Edition | 1of2 Downloaded by guest on September 30, 2021 whereas elimination of p62 suppresses tu- nine deprivation via ADI-PEG20 damaged widespread is the phenotype in arginine- morigenesis by preventing mitochondrial mitochondria, reducing their oxygen con- dependent cancers, and is it restricted to and genomic damage that is linked to oxi- sumption rate and the mitochondrial mem- arginine alone? Second, how can the process dative stress (17). brane potential, consistent with prior results be measured in vivo? The study was re- It is has been argued that many of the in malignant mesothelioma cell lines (10). stricted to cancer cell lines and thus more techniques and tool compounds lack speci- definitive conclusions regarding the role of ficity in the analysis of autophagic cell death, New data by Changou et chromatophagy must await studies per- and that progress will depend on using novel al. highlight that there formed in patients treated with arginine- methodologies and modulating multiple Atg loweringagents.Third,whichagentswillbe members throughout the process of cell death is still much to learn most effective at potentiating the process and (21). The biphasic autophagic response re- about the effects of ported by Changou et al. (16) in arginine- arginine in cancer cells. how should these be sequenced with conven- depleted tumor cells, namely an early prosur- tional as well as newer anticancer agents? vival stage (within 24–48 h) followed by Because it is known that arginine depriva- Indeed, work performed several decades ago delayed chromatophagy (beyond 48 h) linked tion induces free radical formation and showed that L-asparaginase, the first amino to cell death opens up new areas of study for peroxynitrite stress, further studies are acid depletor, had limited clinical utility as the field of amino acid deprivation (Fig. 1). warranted in this area, particularly in rela- a monotherapy in the treatment of acute The formation of giant autophagosomes se- tion to arginine-dependent cancer cells (22). lymphoblastic leukemia (2, 9). It was not un- questering leaked nuclear DNA in association It is also possible that ammonia, a degrada- til it was incorporated into multimodality with chromatin proteins following arginine tion product of ADI-PEG20 (the other being regimens that the impact of L-asparaginase withdrawal is striking—20-fold the size of citrulline, which is readily measured as was observed in the cure of malignant dis- standard vacuoles—and suggestive of a regu- a pharmacodynamic biomarker), may have ease. Fourth, will antioxidants suppress chro- lated process. Changou et al. (16) supple- an additional modulatory role on the pro- matophagy and reduce the effectiveness of mented 4D fluorescence microscopic work cess of autophagy. Recent work has shown arginine deprivation therapy? Fifth, how will that ammonia is released following gluta- with transmission electron microscopic stud- the chromatophagy play out within the host- ies, revealing giant autophagosomes fused to minolysis in tumor cells and activates auto- tumor arginine metabolome? Understanding the nuclear membrane. Moreover, there was phagy (17). Thus, excess nitrogen-sensing the autophagic mechanisms of resistance and a specific lack of lamin A/C proteins, which mechanisms involving peroxynitrite or am- cell death of arginine deprivation is gaining maintain nuclear membrane integrity, thus monia may play a key role in mediating ADI- pointing to chromatophagy as a dynamic induced autophagy. momentum. Integrating this data into the process involved in capturing the leaked More broadly, there remain several unan- hardwiring of the arginine metabolome in DNA. Notably, Atg5 and Beclin-1 short-hair- swered questions with regard to ADI-induced cancer cells will be crucial to the success of pin RNAs suppressed the giant autophago- chromatophagy that are likely to impact on combination therapies that exploit arginine somes, arguing for a major role for these the success of arginine-depleting agents in starvation for a wide spectrum of hard-to- proteins in modulating sensitivity to ADI- the treatment of malignant disease. First, how treat malignancies. PEG20. Similarly, the DNA leakage and chromatophagy was inhibited using the autophagy suppressors 3-methyladenine (via 1 Hanahan D, Weinberg RA (2011) Hallmarks of cancer: The next 12 Huang HY, et al. (2013) ASS1 as a novel tumor suppressor gene generation. 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