Current and upcoming mitochondrial targets for cancer therapy.pdf

Accepted ManuscriptTitle:Current and upcoming mitochondrial targets for cancertherapyAuthors:Hyoung Kyu Kim,Yeon Hee Noh,Bernd Nilius,Kyung Soo Ko,Byoung Doo Rhee,Nari Kim,Jin HanPII:S1044-579X(17)30157-8DOI:http:/dx.doi.org/doi:10.1016/j.semcancer.2017.06.006Reference:YSCBI 1347To appear in:Seminars in Cancer BiologyReceived date:16-11-2016Revised date:1-6-2017Accepted date:9-6-2017Pleasecitethisarticleas:KimHyoungKyu,NohYeonHee,NiliusBernd,Ko Kyung Soo,Rhee Byoung Doo,Kim Nari,Han Jin.Current andupcoming mitochondrial targets for cancer therapy.Seminars in Cancer Biologyhttp:/dx.doi.org/10.1016/j.semcancer.2017.06.006This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.Themanuscriptwillundergocopyediting,typesetting,andreviewoftheresultingproofbefore it is published in its final form.Please note that during the production processerrors may be discovered which could affect the content,and all legal disclaimers thatapply to the journal pertain.1 Current and upcoming mitochondrial targets for cancer therapy Hyoung Kyu Kim1,2,Yeon Hee Noh1,Bernd Nilius3,Kyung Soo Ko1,Byoung Doo Rhee1,Nari Kim1,and Jin Han1*1National Research Laboratory for Mitochondrial Signaling,Department of Physiology,Department of Health Sciences and Technology,BK21 plus Project Team,College of Medicine,Cardiovascular and Metabolic Disease Center,Inje University,Busan,Korea 2Department of Integrated Biomedical Science,College of Medicine,Inje University,Busan,Korea 3KU Leuven,Department Cell Mol Medicine,Leuven,3000,Belgium *Corresponding author:Jin Han(e-mail:phyhanjinje.ac.kr),National Research Laboratory for Mitochondrial Signaling,Department of Physiology,Cardiovascular and Metabolic Disease Center,College of Medicine,Inje University,Bokji-ro 75,Busanjin-gu,Busan 47392,Korea.(Tel)82-51-890-6727,(Fax)82-51-894-5714 Abstract Mitochondria are essential intracellular organelles that regulate energy metabolism,cell death,and signaling pathways that are important for cell proliferation and differentiation.Therefore,mitochondria are fundamentally implicated in cancer biology,including initiation,growth,metastasis,relapse,and acquired drug resistance.Based on these implications,mitochondria have been proposed as a major therapeutic target for cancer treatment.In addition to classical view of mitochondria in cancer biology,recent studies found novel pathophysiological roles of mitochondria in cancer.In this review,we introduce recent concepts of mitochondrial roles in cancer biology including mitochondrial DNA mutation and epigenetic modulation,energy metabolism reprogramming,mitochondrial channels,involvement in metastasis and drug resistance,and cancer stem cells.We also discuss the role of mitochondria in emerging cancer therapeutic strategies,especially cancer immunotherapy and CRISPR-Cas9 system gene therapy.2 Abbreviations:ETC:electron transport complexes,ROS/RNS:reactive oxygen or nitrogen spices,m:mitochondrial inner membrane potential,TCA:TriCarboxylic Acid Cycle,MPTP:mitochondrial permeability transition pore,mtDNA:mitochondrial DNA,ND5:NADH dehydrogenase 5,mtDNMT1:mtDNA methyltransferase,SAM:S-adenosyl-L-methionine,IDH:Isocitrate dehydrogenase,SDH:succinate dehydrogenase,FH:fumarate hydratase,GSH:glutathione,HIF-1:hypoxia-inducible transcription factor-1,KEAP1:Kelch-like ECH-associated protein 1,NRF2:nuclear farter(erythroid-derived 2)-like 2,PDC:pyruvate dehydrogenase complex,PDHE1:PDC E1 subunit,PDK1,2,3 and 4:pyruvate dehydrogenase kinase 1,2,3 or 4,PDP1 and 2:pyruvate dehydrogenase phosphatases 1 or 2,VDAC:voltage dependent anion channel,ANT:adenine nucleotide translocator,CypD:cyclophilin D,MAC:mitochondrial apoptosis-induced channel,mitoKv1.3:mitochondrial voltage-gated potassium channel,MitoIKCa/KCa3.1:mitochondrial intermediated conductance calcium activated potassium channel,BKCa/KCa1.1:large conductance calcium activated potassium channel,SKCa/KCa3.1:small conductance calcium activated potassium channel,mitoKATP:Mitochondrial ATP-sensitive potassium channel,TASK-3:TWIK-related Acid-sensitive K+channel-3,KCNK9,UCP:uncoupling proteins,MCU:Mitochondria Ca2+uniporter,TNBC:triple-negative breast cancer,Mrs2:mitochondrial Mg2+channel,TOM/TIM:translocase of outer/inner membrane,PGC1-:peroxisome proliferator-activated receptor gamma,coactivator 1 alpha,CSC:cancer stem cell,PRX3:peroxiredoxin 3,PD-1:programmed death-1,CTLA-4:cytotoxic T lymphocyte antigen 4,LAG-3:lymphocyte activation gene 3,Tim-3:T cell immunoglobulin and mucin-containing gene 3,CRISPRCas9:clustered regularly interspaced short palindromic repeatsCRISPR-associated 9,CLIC4:chloride intracellular channel 4.Keywords:mitochondria,mitochondria channels,cancer stem cell,cancer immunotherapy,CRISPR-Cas9 1.Introduction Cancer is a highly heterogeneous disease encompassing more than 200 types,many of which are organ or tissue specific.Otto Warburg showed that cancers acquire an uncommon ability to take up glucose and ferment it into lactate in the presence of oxygen,termed the Warburg effect 1.After these early observations,Hanahan and Weinberg suggested a number of common characteristics of cancer 2.These hallmarks included 1)sustained proliferative signals,2)evasion of growth suppression,3)activation of metastasis and invasion,4)replicative immortality,5)induction of angiogenesis,and 6)ability to resist cell death.Recently,four new cancer hallmarks were proposed,3 including 1)avoidance of immune destruction,2)inflammation that promotes tumor formation,3)genome instability,and 4)deregulation of cellular energetics 3.From the early observations of Warburg to the very recent studies,mitochondria,the subcellular organelles primarily responsible for energy production,have taken a central position in our understanding of cancer biology,including tumorigenesis,metastasis,drug resistance,and even the development of different types of cancer therapies 4.Mitochondria have unique features that make them very different from other organelles.Structurally,mitochondria have a double lipid membrane containing various types of membrane proteins including ion channels,transporters,receptors,and electron transport complexes(ETC).The central role of mitochondria is to produce energy,and their highly organized and sophisticated ETC and ATP synthesis system produces a large amount of the bioenergy molecule ATP(40 kg/day in adult humans)very effectively 5.In addition to ATP generation,the mitochondrial ETC is also the major site of reactive oxygen or nitrogen spices(ROS/RNS)production.ROS,when in an optimal concentration range,is an essential signaling molecule regulating cell proliferation,differentiation,inflammation,and hormone synthesis.However,excessive ROS production over the cells antioxidant capacity induces rapid oxidation of various macromolecules including proteins,lipids,and DNA in the nucleus and mitochondria 6.Mitochondrial ion channels transmit various electrical signals via mobilization of Ca2+,Na+,and K+ions,which regulates mitochondrial inner membrane potential(m),TCA cycle activity and ATP production,mitochondrial permeability transition pore(MPTP)opening,and cross-talk between the inside and outside of the mitochondria 7,8.The human mitochondrial matrix contains circular,double-stranded,covalently closed DNA encoding 13 ETC proteins,two ribosomal RNAs,22 transfer RNAs,and a peptide called humanin 9.A set of orchestrated mitochondria functional modifications are implicated in cancer biology,rather than any single change in function.These changes include a shift of energy production mechanisms,increased ROS production and equivalently increased antioxidant activity,disruption of apoptotic signaling,and increased mtDNA mutation 4,10-12.This review will summarize the essential role of mitochondria in various aspects of cancer biology including tumorigenesis,metastasis,drug resistance,cancer stem cell biology,and novel therapies.2.Mitochondrial DNA mutation and epigenetics in tumorigenesis mtDNA was believed to be fragile to oxidative stress due to the lack of a DNA recovery system in the mitochondria like the system that exists in the nucleus 13,14.However,recent studies revealed that mitochondria also have their own DNA repair system 15,16.Nevertheless,the proximity to the ETC and major ROS production sites exposes mtDNA to ROS,causing a relatively high rate of mtDNA 4 mutation accumulation 17.mtDNA mutation and the subsequent dysfunction of the encoded ETC proteins have been suggested as causes of aging and age-linked diseases 18,19.Accumulation of mtDNA mutations occurs in most types of cancers 20-22(Figure 1).Are those mtDNA mutations just passengers of tumorigenesis?A study of the colorectal cancer specific mtDNA mutation in complex I subunit 5(NADH dehydrogenase 5,ND5)demonstrated that the mutation gradually decreases oxidative phosphorylation but increases glucose-dependent lactate production and enhances tumor growth 23.Furthermore,the ND5 mutation-induced complex I dysfunction activates the Akt signaling pathway to enhance tumorigenesis 24.These results suggest mutated mtDNA genes are not just innocent victims,but rather a major initiation factor in tumorigenesis.Developments in epigenetic analyses have made it possible to examine the epigenetic regulation of mtDNA in cancer.Epigenetic modifications of mtDNA include methylation and hydroxymethylation 25.mtDNA methylation is executed by mtDNA methyltransferase(mtDNMT1)in the presence of methyl donor S-adenosyl-L-methionine(SAM),which is synthesized in the cytosol 25.mtDNMT1 expression is upregulated by mitochondrial biogenesis-modulating transcription factors including NRF1 and PGC1,and by loss of the tumor suppressor p53 26.The production of SAM is regulated by mitochondrial ATP synthesis and folate metabolism 27.Methylation occurs at the displacement loop(D-loop)region of mtDNA that regulate expression of mitochondria DNA encoded genes.D-loop demethylation is highly correlated with NADH dehydrogenase 2 expression and both are significantly increased in colorectal cancer 28.In contrast,hypermethylation of mtDNA accompanied a reduced mtDNA copy number,in gastric cancer 29.Based on the accumulated data,mtDNA methylation was suggested as a next-generation biomarker of various diseases including cancer,neurodegenerative diseases,and aging-related degenerative diseases 27.Diagnosis of mtDNA copy number is already widely used as biomarker for various cancers,reflecting the respiratory function,immune response,and cell-cycle modulation of the cancer cells,and could also be an effective therapeutic marker for cancer treatment 30.Collectively,mutation and epigenetic regulation of mtDNA could be major drivers of tumorigenesis,and thus,potential targets for cancer therapy.3.Mitochondrial metabolic reprograming in cancers In the cancer cell,the activity of TCA cycle proteins is significantly altered by mutation of nuclear DNA-encoded TCA cycle genes including isocitrate dehydrogenase(IDH),succinate dehydrogenase(SDH),and fumarate hydratase(FH)31-33.Cancer cells effectively used the TCA cycle during tumorigenesis and survival under stress 34.The detailed mechanisms and effects on cancer biology of the modulation of key TCA cycle enzymes were well reviewed previously 35.Briefly,IDH mutation inhibits the conversion of isocitrate to-ketoglutamate,and increases the level of oncometabolite d-2-hydorxylglutarate,which enhances the formation and malignant progression of gliomas 36.IDH 5 mutation also decreases glutathione(GSH)and NADH levels,resulting in ROS accumulation and oxidative stress-induced DNA mutation which accelerate carcinogenesis(Figure 2a)34,35.The mutation of the SDH gene in hereditary paraganglioma and pheochromocytoma represses the ETC II of the mitochondrial respiratory chain and activates the hypoxia-inducible transcription factor-1 (HIF-1)signaling pathway,leading to tumor growth and vascularization(Figure 2b)37.Upon HIF-1 activation,mutation of SDH results in succinate accumulation,inhibiting the activity of prolyl hydroxylase(PHD).PHD negatively regulates HIF-1 through degradation process under normoxic conditions,further facilitating HIF-1-induced tumorigenesis 38.In addition,accumulated succinate increases ROS generation and oncogenic signaling pathways including those that promote angiogenesis,metastasis,and glycolysis(Figure 2b)34,35.Mutation or depletion of FH suppresses enzymatic conversion of fumarate to malate,resulting in an accumulation of fumarate,which reduces GSH to produce succinated glutathione,an oncometabolite that enhances tumorigenesis 39.Fumarate increases dissociation of Kelch-like ECH-associated protein 1(KEAP1)from nuclear factor(erythroid-derived 2)-like 2(Nrf2),causing the translocation of Nrf2 into the nucleus where it promotes transcription of antioxidant and oncogenic genes(Figure 2c)34,35,40.The TCA cycle provides a useful pool of building blocks to synthesize macromolecules(e.g.,nucleotides,lipids,amino acids)in cancer cells.A major intermediate of the TCA cycle,citrate,is exported to the cytosol and transformed into acetyl-CoA for lipid synthesis or to oxaloacetate/aspartate for nucleotide synthesis 41,42.In the process,cancer cells increase their uptake of glutamine,an alternative source of TCA intermediates and glutathione,to provide building blocks and attenuate oxidative stress.Glutamine metabolism is widely involved in many aspects of cancer biology,including cell proliferation,ATP production,ROS regulation,and stress responses.The importance of glutamine metabolism and its therapeutic potential are well reviewed in a recent article by Altman et al.41.Pyruvate dehydrogenase complex(PDC)is a molecular linker that connect the glycolysis pathway to the TCA cycle through its conversion of pyruvate to acetyl-CoA via pyruvate decarboxylation.The activity of PDC is mainly regulated by the phosphorylation states of its E1 subunit(PDHE1),which are controlled by four pyruvate dehydrogenase kinases(PDK1,-2,-3,and-4)and two pyruvate dehydrogenase phosphatases(PDP1 and-2).There are a number of phosphorylation sites on PDHE1,and when phosphorylated by PDKs,PDC and TCA activity are suppressed.In various cancer types,but not all,the activities of various PDK isoforms were upregulated,inhibiting PDC,the TCA cycle,and mitochondrial respiration(Figure 2d)43.At the transcription level,oncogenes including c-Myc and HIF-1 upregulate expression of PDK1 and-3,while tumor suppressor p53 negatively regulates PDK2.It is known that PDK activity is sensitive to pH changes.Altered cancer cell metabolismincluding decreased respiration,increased glycolysis,and increased glutaminolysiscan boost production of lactate,protons and carbon dioxide,shifting the intracellular environment toward an acidic pH and modulating PDK activity(Figure 2d)43.Some other types of 6 cancer,including breast,ovarian,lung,and co