ONC201 kills breast cancer cells in vitro by targeting mitochondria

We report a novel mechanism of action of ONC201 as a mitochondria-targeting drug in cancer cells. ONC201 was originally identified as a small molecule that induces transcription of TNF-related apoptosis-inducing ligand (TRAIL) and subsequently kills cancer cells by activating TRAIL death receptors. In this study, we examined ONC201 toxicity on multiple human breast and endometrial cancer cell lines. ONC201 attenuated cell viability in all cancer cell lines tested. Unexpectedly, ONC201 toxicity was not dependent on either TRAIL receptors nor caspases. Time-lapse live cell imaging revealed that ONC201 induces cell membrane ballooning followed by rupture, distinct from the morphology of cells undergoing apoptosis. Further investigation found that ONC201 induces phosphorylation of AMP-dependent kinase and ATP loss. Cytotoxicity and ATP depletion were significantly enhanced in the absence of glucose, suggesting that ONC201 targets mitochondrial respiration. Further analysis indicated that ONC201 indirectly inhibits mitochondrial respiration. Confocal and electron microscopic analysis demonstrated that ONC201 triggers mitochondrial structural damage and functional impairment. Moreover, ONC201 decreased mitochondrial DNA (mtDNA). RNAseq analysis revealed that ONC201 suppresses expression of multiple mtDNA-encoded genes and nuclear-encoded mitochondrial genes involved in oxidative phosphorylation and other mitochondrial functions. Importantly, fumarate hydratase deficient cancer cells and multiple cancer cell lines with reduced amounts of mtDNA were resistant to ONC201. These results indicate that cells not dependent on mitochondrial respiration are ONC201-resistant. Our data demonstrate that ONC201 kills cancer cells by disrupting mitochondrial function and further suggests that cancer cells that are dependent on glycolysis will be resistant to ONC201.

[1]  Jaak Vilo,et al.  ClustVis: a web tool for visualizing clustering of multivariate data using Principal Component Analysis and heatmap , 2015, Nucleic Acids Res..

[2]  R. Paules,et al.  An integrated stress response regulates amino acid metabolism and resistance to oxidative stress. , 2003, Molecular cell.

[3]  J. Nunnari,et al.  Mitochondrial form and function , 2014, Nature.

[4]  A. Garnier,et al.  Transcriptional control of mitochondrial biogenesis: the central role of PGC-1alpha. , 2008, Cardiovascular research.

[5]  R. Scarpulla,et al.  PGC-1-Related Coactivator, a Novel, Serum-Inducible Coactivator of Nuclear Respiratory Factor 1-Dependent Transcription in Mammalian Cells , 2001, Molecular and Cellular Biology.

[6]  Mark J. Smyth,et al.  The TRAIL apoptotic pathway in cancer onset, progression and therapy , 2008, Nature Reviews Cancer.

[7]  J. Locasale,et al.  The Warburg Effect: How Does it Benefit Cancer Cells? , 2016, Trends in biochemical sciences.

[8]  S. Joshi,et al.  ATP synthase complex from bovine heart mitochondria: the oligomycin sensitivity conferring protein is essential for dicyclohexyl carbodiimide-sensitive ATPase. , 1991, Biochimica et biophysica acta.

[9]  W. Reinhold,et al.  Using CellMiner 1.6 for Systems Pharmacology and Genomic Analysis of the NCI-60 , 2015, Clinical Cancer Research.

[10]  R. McPherson,et al.  Galactose Enhances Oxidative Metabolism and Reveals Mitochondrial Dysfunction in Human Primary Muscle Cells , 2011, PloS one.

[11]  J. Auwerx,et al.  The mitochondrial unfolded protein response, a conserved stress response pathway with implications in health and disease , 2014, Journal of Experimental Biology.

[12]  John Calvin Reed,et al.  Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities , 2008, Nature Reviews Drug Discovery.

[13]  R. Candau,et al.  The role of AMP-activated protein kinase in the coordination of skeletal muscle turnover and energy homeostasis. , 2012, American journal of physiology. Cell physiology.

[14]  Dongchon Kang,et al.  Frequent truncating mutation of TFAM induces mitochondrial DNA depletion and apoptotic resistance in microsatellite-unstable colorectal cancer. , 2011, Cancer research.

[15]  E. Messaris,et al.  Dual Inactivation of Akt and ERK by TIC10 Signals Foxo3a Nuclear Translocation, TRAIL Gene Induction, and Potent Antitumor Effects , 2013, Science Translational Medicine.

[16]  H. Jacobs,et al.  Alterations to the expression level of mitochondrial transcription factor A, TFAM, modify the mode of mitochondrial DNA replication in cultured human cells , 2006, Nucleic acids research.

[17]  W. El-Deiry,et al.  ONC201 kills solid tumor cells by triggering an integrated stress response dependent on ATF4 activation by specific eIF2α kinases , 2016, Science Signaling.

[18]  G. Barsh,et al.  Mitochondrial transcription factor A is necessary for mtDNA maintance and embryogenesis in mice , 1998, Nature Genetics.

[19]  R. DiPaola,et al.  First-in-Human Clinical Trial of Oral ONC201 in Patients with Refractory Solid Tumors , 2017, Clinical Cancer Research.

[20]  John Calvin Reed,et al.  Endoplasmic reticulum stress: cell life and death decisions. , 2005, The Journal of clinical investigation.

[21]  R. Gilkerson,et al.  The mitochondrial nucleoid: integrating mitochondrial DNA into cellular homeostasis. , 2013, Cold Spring Harbor perspectives in biology.

[22]  P. Fortina,et al.  The reverse Warburg effect: Aerobic glycolysis in cancer associated fibroblasts and the tumor stroma , 2009, Cell cycle.

[23]  W. Linehan,et al.  UOK 262 cell line, fumarate hydratase deficient (FH-/FH-) hereditary leiomyomatosis renal cell carcinoma: in vitro and in vivo model of an aberrant energy metabolic pathway in human cancer. , 2010, Cancer genetics and cytogenetics.

[24]  G. Attardi,et al.  The mtDNA‐encoded ND6 subunit of mitochondrial NADH dehydrogenase is essential for the assembly of the membrane arm and the respiratory function of the enzyme , 1998, The EMBO journal.

[25]  Mila Ljujic,et al.  The integrated stress response , 2016, EMBO reports.

[26]  R. Scarpulla,et al.  PGC-1-Related Coactivator: Immediate Early Expression and Characterization of a CREB/NRF-1 Binding Domain Associated with Cytochrome c Promoter Occupancy and Respiratory Growth , 2006, Molecular and Cellular Biology.

[27]  D. Wallace,et al.  Nonviability of cells with oxidative defects in galactose medium: a screening test for affected patient fibroblasts. , 1992, Biochemical medicine and metabolic biology.

[28]  F. Urano,et al.  Compartment-specific perturbation of protein handling activates genes encoding mitochondrial chaperones , 2004, Journal of Cell Science.

[29]  P. Dennis,et al.  Down-regulation of the erbB-2 receptor by trastuzumab (herceptin) enhances tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in breast and ovarian cancer cell lines that overexpress erbB-2. , 2001, Cancer research.

[30]  W. El-Deiry,et al.  ONC201 Demonstrates Antitumor Effects in Both Triple-Negative and Non–Triple-Negative Breast Cancers through TRAIL-Dependent and TRAIL-Independent Mechanisms , 2017, Molecular Cancer Therapeutics.

[31]  P. Dimroth,et al.  Essentials for ATP synthesis by F1F0 ATP synthases. , 2009, Annual review of biochemistry.

[32]  D. Hardie,et al.  AMPK: a nutrient and energy sensor that maintains energy homeostasis , 2012, Nature Reviews Molecular Cell Biology.

[33]  M. B. Jensen,et al.  Mitochondrial proteostasis in the control of aging and longevity. , 2014, Cell metabolism.

[34]  Rodrigue Rossignol,et al.  Energy Substrate Modulates Mitochondrial Structure and Oxidative Capacity in Cancer Cells , 2004, Cancer Research.

[35]  P. Holland.  Death receptor agonist therapies for cancer, which is the right TRAIL? , 2014, Cytokine & growth factor reviews.

[36]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[37]  K. Inoki,et al.  AMPK and mTOR in cellular energy homeostasis and drug targets. , 2012, Annual review of pharmacology and toxicology.

[38]  Kjell Hultenby,et al.  Mitochondrial transcription factor A regulates mtDNA copy number in mammals. , 2004, Human molecular genetics.

[39]  W. Huber,et al.  Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2 , 2014, Genome Biology.

[40]  P. Meltzer,et al.  TRAIL induces apoptosis in triple-negative breast cancer cells with a mesenchymal phenotype , 2008, Breast Cancer Research and Treatment.

[41]  Yvonne Will,et al.  Circumventing the Crabtree effect: replacing media glucose with galactose increases susceptibility of HepG2 cells to mitochondrial toxicants. , 2007, Toxicological sciences : an official journal of the Society of Toxicology.

[42]  W. El-Deiry,et al.  TRAIL receptor signaling and therapeutics , 2010, Expert opinion on therapeutic targets.

[43]  T. Kuwana,et al.  Chemical inhibition of the mitochondrial division dynamin reveals its role in Bax/Bak-dependent mitochondrial outer membrane permeabilization. , 2008, Developmental cell.

[44]  M. Owen,et al.  Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. , 2000, The Biochemical journal.

[45]  M. Nau,et al.  Chemotherapy augments TRAIL-induced apoptosis in breast cell lines. , 1999, Cancer research.

[46]  S. Lipkowitz,et al.  ONC201: Stressing tumors to death , 2016, Science Signaling.

[47]  P. Wen,et al.  A phase 2 study of the first imipridone ONC201, a selective DRD2 antagonist for oncology, administered every three weeks in recurrent glioblastoma , 2017, Oncotarget.

[48]  Pablo Tamayo,et al.  Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[49]  Lawrence Buja,et al.  Oncosis: an important non-apoptotic mode of cell death. , 2012, Experimental and molecular pathology.

[50]  W. El-Deiry,et al.  Identification of TRAIL-inducing compounds highlights small molecule ONC201/TIC10 as a unique anti-cancer agent that activates the TRAIL pathway , 2015, Molecular Cancer.

[51]  M. Palacín,et al.  Mitochondrial fusion proteins: dual regulators of morphology and metabolism. , 2010, Seminars in cell & developmental biology.

[52]  Kenneth M. Yamada,et al.  Direct comparisons of the morphology, migration, cell adhesions, and actin cytoskeleton of fibroblasts in four different three-dimensional extracellular matrices. , 2011, Tissue engineering. Part A.

[53]  A. Ashkenazi.  Targeting the extrinsic apoptotic pathway in cancer: lessons learned and future directions. , 2015, The Journal of clinical investigation.

[54]  J. Wagner,et al.  Reverse Warburg effect in a patient with aggressive B-cell lymphoma: is lactic acidosis a paraneoplastic syndrome? , 2013, Seminars in oncology.

[55]  R. Scarpulla.  Transcriptional paradigms in mammalian mitochondrial biogenesis and function. , 2008, Physiological reviews.

[56]  Gen Sheng Wu,et al.  ONC201 activates ER stress to inhibit the growth of triple-negative breast cancer cells , 2017, Oncotarget.

[57]  Andrea Glasauer,et al.  Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis , 2014, eLife.

[58]  Pablo E. Morales,et al.  Mitochondrial metabolism and the control of vascular smooth muscle cell proliferation , 2014, Front. Cell Dev. Biol..

[59]  E. D. de Vries,et al.  Translating TRAIL-receptor targeting agents to the clinic. , 2013, Cancer letters.

[60]  K. Hashiguchi,et al.  Establishment of human cell lines lacking mitochondrial DNA. , 2009, Methods in molecular biology.

[61]  Colin N. Dewey,et al.  RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome , 2011, BMC Bioinformatics.

[62]  Cole M. Haynes,et al.  Metabolism and the UPR(mt). , 2016, Molecular cell.

[63]  G. Salvesen,et al.  Regulated cell death: signaling and mechanisms. , 2014, Annual review of cell and developmental biology.

[64]  F. Sotgia,et al.  Using the “reverse Warburg effect” to identify high-risk breast cancer patients , 2012, Cell cycle.

[65]  C. Koumenis,et al.  Cell intrinsic and extrinsic activators of the unfolded protein response in cancer: Mechanisms and targets for therapy. , 2015, Seminars in cancer biology.

[66]  J. Nunnari,et al.  Mitochondria: In Sickness and in Health , 2012, Cell.

[67]  Michael L. Wang,et al.  ATF4 induction through an atypical integrated stress response to ONC201 triggers p53-independent apoptosis in hematological malignancies , 2016, Science Signaling.

[68]  U. Matthey,et al.  Crucial role of the membrane potential for ATP synthesis by F(1)F(o) ATP synthases. , 2000, The Journal of experimental biology.

[69]  Chris Sander,et al.  rcellminer: exploring molecular profiles and drug response of the NCI-60 cell lines in R , 2016, Bioinform..

[70]  J. Herman,et al.  Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[71]  W. El-Deiry,et al.  ONC201 induces cell death in pediatric non-Hodgkin's lymphoma cells , 2015, Cell cycle.