Abstract
In contrast to the “Warburg effect” or aerobic glycolysis earlier generalized as a phenomenon in cancer cells, more and more recent evidence indicates that functional mitochondria are pivotal for ensuring the energy supply of cancer cells. Here, we report that cancer cells with reduced autophagy-related protein 12 (ATG12) expression undergo an oncotic cell death, a phenotype distinct from that seen in ATG5-deficient cells described before. In addition, using untargeted metabolomics with ATG12-deficient cancer cells, we observed a global reduction in cellular bioenergetic pathways, such as β-oxidation (FAO), glycolysis, and tricarboxylic acid cycle activity, as well as a decrease in mitochondrial respiration as monitored with Seahorse experiments. Analyzing the biogenesis of mitochondria by quantifying mitochondrial DNA content together with several mitochondrion-localizing proteins indicated a reduction in mitochondrial biogenesis in ATG12-deficient cancer cells, which also showed reduced hexokinase II expression and the upregulation of uncoupling protein 2. ATG12, which we observed in normal cells to be partially localized in mitochondria, is upregulated in multiple types of solid tumors in comparison with normal tissues. Strikingly, mouse xenografts of ATG12-deficient cells grew significantly slower as compared with vector control cells. Collectively, our work has revealed a previously unreported role for ATG12 in regulating mitochondrial biogenesis and cellular energy metabolism and points up an essential role for mitochondria as a failsafe mechanism in the growth and survival of glycolysis-dependent cancer cells. Inducing oncosis by imposing an ATG12 deficiency in solid tumors might represent an anticancer therapy preferable to conventional caspase-dependent apoptosis that often leads to undesirable consequences, such as incomplete cancer cell killing and a silencing of the host immune system.
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References
Fulda S. Targeting autophagy for the treatment of cancer. Biol Chem. 2018;399:673–7.
Koyama-Honda I, Itakura E, Fujiwara TK, Mizushima N. Temporal analysis of recruitment of mammalian ATG proteins to the autophagosome formation site. Autophagy. 2013;9:1491–9.
Yousefi S, Perozzo R, Schmid I, Ziemiecki A, Schaffner T, Scapozza L, et al. Calpain-mediated cleavage of Atg5 switches autophagy to apoptosis. Nat Cell Biol. 2006;8:1124–32.
Maskey D, Yousefi S, Schmid I, Zlobec I, Perren A, Friis R, et al. ATG5 is induced by DNA-damaging agents and promotes mitotic catastrophe independent of autophagy. Nat Commun. 2013;4:2130.
Rubinstein AD, Eisenstein M, Ber Y, Bialik S, Kimchi A. The autophagy protein Atg12 associates with antiapoptotic Bcl-2 family members to promote mitochondrial apoptosis. Mol Cell. 2011;44:698–709.
Haller M, Hock AK, Giampazolias E, Oberst A, Green DR, Debnath J, et al. Ubiquitination and proteasomal degradation of ATG12 regulates its proapoptotic activity. Autophagy. 2014;10:2269–78.
Radoshevich L, Murrow L, Chen N, Fernandez E, Roy S, Fung C, et al. ATG12 conjugation to ATG3 regulates mitochondrial homeostasis and cell death. Cell. 2010;142:590–600.
Guo JY, Teng X, Laddha SV, Ma S, Van Nostrand SC, Yang Y, et al. Autophagy provides metabolic substrates to maintain energy charge and nucleotide pools in Ras-driven lung cancer cells. Genes Dev. 2016;30:1704–17.
Shubin AV, Demidyuk IV, Komissarov AA, Rafieva LM, Kostrov SV. Cytoplasmic vacuolization in cell death and survival. Oncotarget. 2016;7:55863–89.
Lin JF, Lin YC, Tsai TF, Chen HE, Chou KY, Hwang TI. Cisplatin induces protective autophagy through activation of BECN1 in human bladder cancer cells. Drug Des Devel Ther. 2017;11:1517–33.
Liu H, He Z, von Rutte T, Yousefi S, Hunger RE, Simon HU. Down-regulation of autophagy-related protein 5 (ATG5) contributes to the pathogenesis of early-stage cutaneous melanoma. Sci Transl Med. 2013;5:202ra123.
Hwang SH, Han BI, Lee M. Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2. J Cell Physiol. 2018;233:506–15.
Rozman S, Yousefi S, Oberson K, Kaufmann T, Benarafa C, Simon HU. The generation of neutrophils in the bone marrow is controlled by autophagy. Cell Death Differ. 2015;22:445–56.
Del Nagro C, Xiao Y, Rangell L, Reichelt M, O'Brien T. Depletion of the central metabolite NAD leads to oncosis-mediated cell death. J Biol Chem. 2014;289:35182–92.
Weerasinghe P, Buja LM. Oncosis: an important non-apoptotic mode of cell death. Exp Mol Pathol. 2012;93:302–8.
St John JC, Amaral A, Bowles E, Oliveira JF, Lloyd R, Freitas M, et al. The analysis of mitochondria and mitochondrial DNA in human embryonic stem cells. Methods Mol Biol. 2006;331:347–74.
Liu H, He Z, April SL, Trefny MP, Rougier JS, Salemi S, et al. Biochemical re-programming of human dermal stem cells to neurons by increasing mitochondrial membrane potential. Cell Death Differ. 2019;26:1048–1061.
Morgan AJ, Jacob R. Ionomycin enhances Ca2+ influx by stimulating store-regulated cation entry and not by a direct action at the plasma membrane. Biochem J. 1994;300:665–72.
Warburg O, Wind F, Negelein E. The metabolism of tumors in the body. J Gen Physiol. 1927;8:519–30.
Potter M, Newport E, Morten KJ. The Warburg effect: 80 years on. Biochem Soc Trans. 2016;44:1499–505.
Hao W, Chang CP, Tsao CC, Xu J. Oligomycin-induced bioenergetic adaptation in cancer cells with heterogeneous bioenergetic organization. J Biol Chem. 2010;285:12647–54.
Gatenby RA, Gillies RJ. Why do cancers have high aerobic glycolysis? Nat Rev Cancer. 2004;4:891–9.
Lis P, Dylag M, Niedzwiecka K, Ko YH, Pedersen PL, Goffeau A, et al. The HK2 dependent "Warburg effect" and mitochondrial oxidative phosphorylation in cancer: targets for effective therapy with 3-bromopyruvate. Molecules. 2016;21:1730.
Ngamsiri P, Watcharasit P, Satayavivad J. Glycogen synthase kinase-3 (GSK3) controls deoxyglucose-induced mitochondrial biogenesis in human neuroblastoma SH-SY5Y cells. Mitochondrion. 2014;14:54–63.
Xu W, Zhu H, Gu M, Luo Q, Ding J, Yao Y, et al. DHTKD1 is essential for mitochondrial biogenesis and function maintenance. FEBS Lett. 2013;587:3587–92.
Malik AN, Shahni R, Rodriguez-de-Ledesma A, Laftah A, Cunningham P. Mitochondrial DNA as a non-invasive biomarker: accurate quantification using real time quantitative PCR without co-amplification of pseudogenes and dilution bias. Biochem Biophys Res Commun. 2011;412:1–7.
Mizushima N, Sugita H, Yoshimori T, Ohsumi Y. A new protein conjugation system in human. The counterpart of the yeast Apg12p conjugation system essential for autophagy. J Biol Chem. 1998;273:33889–92.
Valle A, Oliver J, Roca P. Role of uncoupling proteins in cancer. Cancers. 2010;2:567–91.
Mills EM, Xu D, Fergusson MM, Combs CA, Xu Y, Finkel T. Regulation of cellular oncosis by uncoupling protein 2. J Biol Chem. 2002;277:27385–92.
Azzu V, Brand MD. Degradation of an intramitochondrial protein by the cytosolic proteasome. J Cell Sci. 2010;123(Pt 4):578–85.
Rousset S, Mozo J, Dujardin G, Emre Y, Masscheleyn S, Ricquier D, et al. UCP2 is a mitochondrial transporter with an unusual very short half-life. FEBS Lett. 2007;581:479–82.
Itami N, Shiratsuki S, Shirasuna K, Kuwayama T, Iwata H. Mitochondrial biogenesis and degradation are induced by CCCP treatment of porcine oocytes. Reproduction. 2015;150:97–104.
Schroder B, Wrocklage C, Pan C, Jager R, Kosters B, Schafer H, et al. Integral and associated lysosomal membrane proteins. Traffic. 2007;8:1676–86.
Chang SC, Momburg F, Bhutani N, Goldberg AL. The ER aminopeptidase, ERAP1, trims precursors to lengths of MHC class I peptides by a "molecular ruler" mechanism. Proc Natl Acad Sci USA. 2005;102:17107–12.
Bogenhagen DF, Rousseau D, Burke S. The layered structure of human mitochondrial DNA nucleoids. J Biol Chem. 2008;283:3665–75.
Chan DC. Mitochondria: dynamic organelles in disease, aging, and development. Cell. 2006;125:1241–52.
Liu H, He Z, Simon HU. Targeting autophagy as a potential therapeutic approach for melanoma therapy. Semin Cancer Biol. 2013;23:352–60.
Malhotra R, Warne JP, Salas E, Xu AW, Debnath J. Loss of Atg12, but not Atg5, in pro-opiomelanocortin neurons exacerbates diet-induced obesity. Autophagy. 2015;11:145–54.
Galluzzi L, Morselli E, Kepp O, Vitale I, Rigoni A, Vacchelli E, et al. Mitochondrial gateways to cancer. Mol Asp Med. 2010;31:1–20.
Kelly DP, Scarpulla RC. Transcriptional regulatory circuits controlling mitochondrial biogenesis and function. Genes Dev. 2004;18:357–68.
Twig G, Hyde B, Shirihai OS. Mitochondrial fusion, fission and autophagy as a quality control axis: the bioenergetic view. Biochim Biophys Acta. 2008;1777:1092–7.
Youle RJ, Narendra DP. Mechanisms of mitophagy. Nat Rev Mol Cell Biol. 2011;12:9–14.
Zhang H, Gao P, Fukuda R, Kumar G, Krishnamachary B, Zeller KI, et al. HIF-1 inhibits mitochondrial biogenesis and cellular respiration in VHL-deficient renal cell carcinoma by repression of C-MYC activity. Cancer Cell. 2007;11:407–20.
Ichim G, Lopez J, Ahmed SU, Muthalagu N, Giampazolias E, Delgado ME, et al. Limited mitochondrial permeabilization causes DNA damage and genomic instability in the absence of cell death. Mol Cell. 2015;57:860–72.
Law BY, Mok SW, Chan WK, Xu SW, Wu AG, Yao XJ, et al. Hernandezine, a novel AMPK activator induces autophagic cell death in drug-resistant cancers. Oncotarget. 2016;7:8090–104.
Guan F, Ding Y, Zhang Y, Zhou Y, Li M, Wang C. Curcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Akt degradation. PLoS ONE. 2016;11:e0146553.
Okayama H, Chen C. Calcium phosphate mediated gene transfer into established cell lines. Methods Mol Biol. 1991;7:15–21.
Rindlisbacher B, Schmid C, Geiser T, Bovet C, Funke-Chambour M. Serum metabolic profiling identified a distinct metabolic signature in patients with idiopathic pulmonary fibrosis—a potential biomarker role for LysoPC. Respir Res. 2018;19:7.
Li S, Park Y, Duraisingham S, Strobel FH, Khan N, Soltow QA, et al. Predicting network activity from high throughput metabolomics. PLoS Comput Biol. 2013;9:e1003123.
Tan B, Xiao H, Li F, Zeng L, Yin Y. The profiles of mitochondrial respiration and glycolysis using extracellular flux analysis in porcine enterocyte IPEC-J2. Anim Nutr. 2015;1:239–43.
Phillips NR, Sprouse ML, Roby RK. Simultaneous quantification of mitochondrial DNA copy number and deletion ratio: a multiplex real-time PCR assay. Sci Rep. 2014;4:3887.
Conus S, Perozzo R, Reinheckel T, Peters C, Scapozza L, Yousefi S, et al. Caspase-8 is activated by cathepsin D initiating neutrophil apoptosis during the resolution of inflammation. J Exp Med. 2008;205:685–98.
Shultz LD, Lyons BL, Burzenski LM, Gott B, Chen X, Chaleff S, et al. Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol. 2005;174:6477–89.
Acknowledgements
We thank the Clinical Metabolomics Facility, Center for Laboratory Medicine from the Bern University Hospital (Inselspital Bern) for performing UHPLC-HRMS analysis. We also thank Dr José Alberto Galvάn Hernάndez (Institute of Pathology, University of Bern) for his help in evaluating the pathological images, Jeannine Wagner (Institute of Anatomy, University of Bern) for her assistance in electron microscopy and Michaela Römmele (Department for BioMedical Research, University of Bern) for helping us with the mouse xenograft model. NG and ZF are PhD students of the Graduate School of Cellular and Biomedical Sciences of the University of Bern. Images were acquired on equipment supported by the Microscopy Imaging Centre of the University of Bern. This work was supported by grants from the Swiss National Science Foundation (grant no. 310030_166473 and 310030_184816), Swiss Cancer League (grant no. KFS-3703-08-2015), Stiftung zur Krebsbekämpfung (Zurich) and European Union’s Horizon 2020 research and innovation program (Marie Sklodowska-Curie grant no. 642295; MEL-PLEX).
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Liu, H., He, Z., Germič, N. et al. ATG12 deficiency leads to tumor cell oncosis owing to diminished mitochondrial biogenesis and reduced cellular bioenergetics. Cell Death Differ 27, 1965–1980 (2020). https://doi.org/10.1038/s41418-019-0476-5
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DOI: https://doi.org/10.1038/s41418-019-0476-5
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