Significance Nutritional starvation therapy is under intensive investigation because it provides a potentially lower toxicity with higher specificity than conventional cancer therapy. Autophagy, often triggered by starvation, represents an energy-saving, pro-survival cellular function; however, dysregulated autophagy could also lead to cell death, a process distinct from the classic caspase-dependent apoptosis. This study shows how arginine starvation specifically kills tumor cells by a novel mechanism involving mitochondria dysfunction, reactive oxygen species generation, DNA leakage, and chromatin autophagy, where leaked DNA is captured by giant autophagosomes. These results not only provide insights into the fundamental process of metabolic stress-based cancer therapy but also uncover a new cell-death mechanism.

Abstract Autophagy is the principal catabolic prosurvival pathway during nutritional starvation. However, excessive autophagy could be cytotoxic, contributing to cell death, but its mechanism remains elusive. Arginine starvation has emerged as a potential therapy for several types of cancers, owing to their tumor-selective deficiency of the arginine metabolism. We demonstrated here that arginine depletion by arginine deiminase induces a cytotoxic autophagy in argininosuccinate synthetase (ASS1)-deficient prostate cancer cells. Advanced microscopic analyses of arginine-deprived dying cells revealed a novel phenotype with giant autophagosome formation, nucleus membrane rupture, and histone-associated DNA leakage encaptured by autophagosomes, which we shall refer to as chromatin autophagy, or chromatophagy. In addition, nuclear inner membrane (lamin A/C) underwent localized rearrangement and outer membrane (NUP98) partially fused with autophagosome membrane. Further analysis showed that prolonged arginine depletion impaired mitochondrial oxidative phosphorylation function and depolarized mitochondrial membrane potential. Thus, reactive oxygen species (ROS) production significantly increased in both cytosolic and mitochondrial fractions, presumably leading to DNA damage accumulation. Addition of ROS scavenger N-acetyl cysteine or knockdown of ATG5 or BECLIN1 attenuated the chromatophagy phenotype. Our data uncover an atypical autophagy-related death pathway and suggest that mitochondrial damage is central to linking arginine starvation and chromatophagy in two distinct cellular compartments.

There is considerable evidence that tumor and normal cells differ in their metabolic requirements. The most prominent examples are the addiction of tumor cells to glucose (i.e., Warburg effect) and to glutamine (1⇓–3). Therapeutics based on selective targeting of these metabolic pathways are under intensive investigation. Starvation therapy generally posts an advantage of having lower toxicity than conventional radiation and chemotherapy. In addition to glutamine, the differential requirement of other amino acids by tumor cells also exists and has been exploited in developing amino acid depletion therapy. The choices, however, are limited, because only 11 amino acids are considered semiessential or nonessential. Nevertheless, recent studies showed that starvation of arginine, asparagine, cysteine, leucine, and glutamine seems to provide preferential killing of tumor cells (4⇓⇓⇓⇓–9). Among them, arginine and asparagine depletion probably are the most advanced in amino acid starvation therapies and have reached clinical trials (10, 11).

Argininosuccinate synthetase (ASS1), a rate-limiting enzyme for intracellular arginine synthesis, was found to have reduced expression in many cancer types including prostate cancer (4, 5, 12⇓⇓⇓⇓⇓–18). As a result, prostate cancer cells become “auxotroph” for and addicted to external arginine. Indeed, in recent publications we showed that depletion of arginine effectively induces cell death of castration-resistant prostate cancer cells but not of normal prostate epithelial cells in vitro and in vivo (18, 19). This was made possible by the availability of pegylated arginine deiminase (ADI-PEG20), a recombinant mycoplasma protein (Polaris), which converts arginine to citrulline and effectively removes extracellular arginine. Previous worldwide clinical trials of ADI-PEG20 showed it is well tolerated, and the FDA has recently approved it for phase III clinical trials in hepatocellular carcinoma (10, 20).

ADI-PEG20 treatment of prostate cancer cells was accompanied by profound autophagy and caspase-independent cell death (18, 19). In this paper, we further demonstrated that prolonged ADI-PEG20 treatment leads to DNA leakage and that this leaked DNA, together with histones and other chromatin-associated proteins, was captured by LC3-containing autophagosomes. We shall refer to it as “chromatophagy.” This nuclear leakage phenomenon was associated with compromised mitochondrial oxidative phosphorylation and increased reactive oxygen species (ROS) and accumulated DNA damage. Our studies revealed a previously unidentified cellular death mechanism associated with therapeutic arginine deprivation.

Materials and Methods Microscope Image Acquisition and Image Analysis. For autophagy image acquisition, cells were imaged with wide-field DeltaVision deconvolution microscope (Applied Precision Inc.), equipped with 60×/1.42 N.A. oil-immersion objective lens. Both microscope and camera were controlled by SoftWorX application suite software. Stacks of optical section images, with an image size of 512 × 512 pixels, were collected for all fluorochromes. For live-cell imaging, cells were maintained and visualized by ONIX microfluidic perfusion system (CellASIC) and DeltaVision deconvolution microscope, respectively. For fixed-cell imaging, all target proteins were labeled with appropriate primary antibody (see SI Materials and Methods). Nucleus was visualized with DAPI or DRAQ5 (BioStatus) staining following the manufacturer’s protocol. Lysosomes were labeled with anti-LAMP1 (DSHB) for fixed cells or by LysoTracker Red (Invitrogen) for live cells following the manufacturer’s protocol. Stacks of fluorescence images were deconvolved by using SoftWorX software (Applied Precision Inc.) and later analyzed with VoloCITY software (PerkinElmer). All cell samples were scored for the presence of DNA leakage, and more than one leaked DNA or captured DNA by autophagosomes per cell reflects ADI-induced DNA damage. TEM. The ADI-PEG20–treated CWR22Rv1 cells were collected and loaded into flat specimen holders for high-pressure freezing in an electron microscopy PACT HPF station (Leica Microsystems). The samples were freeze-substituted in acetone containing 0.2% glutaraldehyde and 0.1% uranyl acetone at −90 °C for 72 h and then warmed up gradually to −20 °C (AFS; Leica Microsystems) to complete substitution. After dehydration in ethanol of increasing concentration, followed by a series of Lowicryl–ethanol mixtures, samples were infiltrated with pure Lowicryl resin for 16 h. Finally, the resin polymerization was performed at −50 °C under UV light. The sample block was then trimmed and sectioned with a Leica Ultracut microtome. Sections of 100-nm to 120-nm thickness were collected on copper grids for imaging under a JEOL 1230 electron microscope. The electron micrographs were recorded on a F214CCD camera (TVIPS Gauting). Additional methods, extended data display items, and discussion are available in Supporting Information.

Acknowledgments We thank Drs. Bor-Wen Wu, John Bomalaski, and Wei-Jen Kung (Polaris, Taiwan) for providing high-quality ADI-PEG20. We also thank GeneTech Biotech (Taiwan) for providing access to the DV microscope. This work was supported in part by National Institutes of Health (NIH) Grants CA165263, CA150197, CA150197S1, NHRI 02D1-MMD0H02 (Taiwan), and DOH102-TD-M-111-102001 (to H.-J.K.), Grant CA150197S1 (to C.A.C.), and Grants DE10742 and DE14183 (to D.K.A.). Y.-R.C. was supported by a City of Hope Women’s Cancers Program Award. This work was also supported by an Isabelle J. McDonald Endowment and Department of Defense Grant W81XWH-08-1-385 (to R.J.B.). The Center for Biophotonics Science and Technology is managed by the University of California, Davis, under Cooperative Agreement PHY 0120999 (to F.Y.S.C.). R.H.C. was supported by NIH Grant AI095382 and Discovery Grant UCDG178969.