1、Cancer stem cells:Radioresistance,prediction of radiotherapy outcomeand specific targets for combined treatmentsMechthild Krausea,b,c,d,e,Anna Dubrovskaa,c,e,Annett Lingea,b,c,e,Michael Baumanna,b,c,d,eaGerman Cancer Consortium(DKTK)Dresden,GermanybDept.of Radiation Oncology,Technische Universitt Dr
2、esden,GermanycOncoRay,Faculty of Medicine and University Hospital Carl Gustav Carus,Technische Universitt Dresden,GermanydHelmholtz-Zentrum Dresden-Rossendorf,GermanyeGerman Cancer Research Center(DKFZ)Heidelberg,Germanya b s t r a c ta r t i c l ei n f oArticle history:Received 21 September 2015Rec
3、eived in revised form 5 January 2016Accepted 3 February 2016Available online 12 February 2016Inactivation of cancer stem cells(CSCs)is of utmost importance for tumor cure after radiotherapy.An increasingbodyofevidencecomplieswith a higherradioresistanceofCSCs comparedto themassoftumorcells,supportin
4、gthe useofCSC related biomarkers for prediction of radiotherapy outcome.Treatment individualization strategiesfor patient groups with vastly different risk of recurrence will most likely require application of more than onebiomarker.Specifically,inclusion of established biomarkers like tumor size fo
5、r primary radio(chemo)therapyor human papilloma virus(HPV)infection status in head and neck squamous cell carcinoma seems to be ofvery high relevance.The high heterogeneity of CSC subclones along with changes of the functional behavior ofindividualtumorsundertreatmentunderlinestheimportanceofthesele
6、ctionoftheoptimaltimepoint(s)ofbio-marker evaluation,but also provides a potential therapeutic target for combined treatment approaches withirradiation.2016 The Authors.Published by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http:/creativecommons.org/licenses/by-nc-nd/
7、4.0/).Keywords:Cancer stem cellsRadiotherapyPredictionBiomarkerPrognosisContents1.Introduction.632.Radioresistance determined by cancer stem cells.643.Cancer stem cells as prognostic or predictive biomarkers in patient tumors.664.Importance of further biomarkers for patient stratification.675.Intrat
8、umoral heterogeneity and changes of biomarker profiles during treatment.686.Strategies to enhance efficacy of radiotherapy by targeting cancer stem cells.697.Conclusion.70References.70Advanced Drug Delivery Reviews 109(2017)6373Abbreviations:ABCG2,cATP-binding cassette sub-family G member 2;ALDH1A1,
9、aldehyde dehydrogenase 1A1;ATM,ataxia telangiectasia mutated protein kinase;ATR,ATM-Rad-3-related protein kinase;CD,cluster of differentiation;CSC(s),cancer stem cell(s);Chk,checkpoint kinase;CXCR4,chemokine(CXC motif)receptor 4;DNA,deoxyribonucleic acid;DDR,DNA damage response;DSB,double strand bre
10、aks;DZNep,3-Deazaneplanocin A;EGFR,epidermal growth factor receptor;EMT,epithelialmesenchymal transition;FAK,focal adhesionkinase;GBM,glioblastoma multiforme;HA,hyaluronan;HGFR,hepatocyte growth factor receptor;HMG CoA,3-hydroxy-3-methylglutaryl-coenzyme-A;ECM,extracellular matrix;HIF,hypoxia-induci
11、ble factor;HNSCC,head and neck squamous cell carcinoma;HPV,human papilloma virus;HR,homologous recombination;HRE,hypoxia response element;MGMT,O-6-methylguanine-DNA methyltransferase;NHEJ,non-homologous end joining;PET,positron emission tomography;PI3K,phosphatidylinositide 3-kinases;ROS,reactive ox
12、ygen species;SCL3A2,solute carrier family 3A2;SSB,single strand breaks;TCD50,tumor control dose 50%;TD50,transplantation dose 50%;WNT,wingless and INT-1.This review is part of the Advaced Drug Delivery Reviews theme issue on“Radiotherapy for Cancer:Present and Future”.Corresponding author at:Dept.of
13、 Radiation Oncology,University Hospital C.G.Carus,Technische Universitt Dresden,Fetscherstr,74,01307 Dresden,Germany.Tel.:+49 03514585441;fax:+49 0351 4585716.E-mail address:mechthild.krauseuniklinikum-dresden.de(M.Krause).64http:/dx.doi.org/10.1016/j.addr.2016.02.0020169-409X/2016 The Authors.Publi
14、shed by Elsevier B.V.This is an open access article under the CC BY-NC-ND license(http:/creativecommons.org/licenses/by-nc-nd/4.0/).Contents lists available at ScienceDirectAdvanced Drug Delivery Reviewsjournal homepage: stem cells(CSCs)play a pivotal role in tumor development,progression and recurr
15、ence after treatment.CSCs are defined as tumorcellsthathaveanunlimitedpotentialofcelldivisionandanabilitytore-populate the whole tumor 1.For radiotherapy as well as for other po-tentially curative anti-cancer treatments,this definition implies that allCSCs need to be inactivated in order to permanen
16、tly eradicate a tumor.The long established gold standard assay to determine CSCs is a func-tional assay,i.e.the tumor cell transplantation assay measuring thecell number needed to be injected to cause tumor takes in 50%(TD50)of the experimental animals.Technological advances allow us to identi-fy an
17、d sort tumor cells for CSC-rich and CSC-low subpopulations,e.g.bycell surface marker expression.Such markers can in many cases also beidentified by usingstaining techniques in tumor sections or by geneex-pression arrays,ensuring clinical applicability of such parameters forbiomarker assays.However,l
18、imitations occur from the fact that thresh-old values for staining intensity cannot be determined by direct valida-tion of the stemness of the stained tumors cells and from the generalfact applying to all CSC marker assays,that the marker expression isnot specific to CSCs but just determines cells t
19、hat have a higher likeli-hood to express CSC features.Thus,stemness of cells has still to be val-idated by the functional assays mentioned above 2,3.Becauseoftheirimportancefortumorcurability,CSCscanbeutilizedfor strategies to optimize anti-cancer treatments by their evaluation asmarkers for patient
20、 stratification to specific intensified or de-escalatedtreatment strategies and/or by developing treatment approaches thatspecifically target tumor cells expressing putative CSC markers.2.Radioresistance determined by cancer stem cellsIf other confoundingfactors are constant,the radiation dose to er
21、ad-icate a tumor inversely correlates with the logarithm of the number ofCSCs,i.e.at the same radiotherapy dose tumors with a lower numberof CSCs show higher local control rates compared with tumors withhigher numbers of CSCs.In experimental and clinical data,this leadsto a tumor volume dependence o
22、f the curability of tumors,becausetheabsolutenumber of CSCsis expected toincrease withtumor volume26.However,also densities of CSCs can differ between tumors,againimpacting local tumor control after radiotherapy 6,7.The dependenceof local tumor control from the number of CSCs contained in a tumor is
23、underlinedbythelog-linearcorrelationbetweentheTD50andtheradi-ation dose necessary to permanently cure 50%of the tumors(tumorcontrol dose 50%,TCD50)3,8.This correlation indicates that,eventhough CSCs seem to express a constant plasticity,TCD50 is a valuablecorrelate of the number of CSCs in a given t
24、umor.Nevertheless,someofthedescribedtumormodelswithequalTD50showsignificantdiscor-dance in TCD50 values,suggesting that in addition to the total numberof CSCs in a given tumor,the inherent radiosensitivity of CSCs as wellas other radiobiological parameters may impact local tumor controlafter irradia
25、tion 2,8.Such differences of intrinsic radiosensitivity be-tween colony-forming cells in vitro(clonogenic cells)of tumors withthe same histology have been shown by the variation of the survivingfraction at 2 Gy(SF2)in colony-forming in vitro assays,which in somestudies correlate with clinically obse
26、rved differences in radiosensitivitybetween tumor entities or within the same histology 3,9,10.The samecorrelation between radiosensitivity of clonogenic cells in vitro andtumor radiocurability in vivo exists for tumors of different histologies1114,supporting a correlation between clonogenicity in v
27、itro andstemness in vivo and the importance of intrinsic radiosensitivity ofCSCs for radiocurability of tumors.Of note,such preclinical data onestablished tumor models are of high importance for our todaysunder-standing of the biology of CSC,however,specific questions especiallyconcerningdetailedgen
28、eexpressionsortumortonormaltissueinterac-tions may in many cases require the use of primary xenografts.It is notthe scope of this review to discuss the use of different experimentalmodels in detail.Taking into account that tumors can potentially arise from a singleCSC,a failure of radiation treatmen
29、t might be attributed to the incom-plete eradication of the entire CSC population 3,15.The cure rate of ir-radiation results from its ability to induce DNA damage in tumor tissuethrough production of the water-derived reactive oxygen species or bydirect ionization of the DNA molecules 16.At the cell
30、ular level,DNAdamagehaslongandshorttermconsequences.Anacuteeffectisrelatedto thedisturbanceof DNAmetabolismsuchasDNA replication andRNAtranscription.Based on the status of DNA repair and the nature of theDNAlesions,theoutcomeofDNAinjurycanbetemporaryorpermanentcell arrest,immediate or delayed cell d
31、eath or mutagenesis of the sur-viving cells,which in turn might lead to genomic instability and subse-quently results in tumor development in a long term perspective 17.The biological consequences of irradiation leading to cell death arehighly influenced by the activation of the DNA damage response(
32、DDR)mechanisms.Among the various types of DNA damages pro-duced by ionizing irradiation,i.e.single strand breaks(SSBs),doublestrand breaks(DSBs),damaged nucleotide bases or abasic sites,DSBsrepresent the principal lesions that might lead to cell death if not ade-quately repaired 16.Both normal and t
33、umor cells can repair DSBsby either error-free homology-directed recombination(HR)or error-prone non-homologous end joining(NHEJ)mechanisms which are act-ing in an overlapping and complementary manner 18.In addition tothe activation of DNA repair process,DNA damage induces checkpointkinase signaling
34、 pathways such as ataxia telangiectasia mutated(ATM)-checkpoint kinase 2(Chk2)and ATM-Rad3-related(ATR)-checkpoint kinase(Chk1)which delay cell cycle progression in orderto allow DNA repair 18.When DNA damage is beyond the cell repaircapacity,proteins of DDR signaling mediate cellular death or loss
35、ofreproductive capacity through activation of different pathways such asapoptosis,senescence or mitotic catastrophe 19.Therefore,the effica-cy of the DNA repair machinery activated by the DNA damage signalingnetwork is critical and determines cell death or repair.A high DNA re-pair capacity has been
36、 described for CSC populations in differenttumor entities including glioblastoma,prostate,lungand breast cancersand mainly attributed to the activation of the ATR-Chk1 and ATM-Chk2signaling pathways 2030,as summarized in Table 1.Radiation-induced cell death occurs not only by a direct energytransfer
37、 to the DNA but,in case of conventional photon radiotherapy,mainly asa result of generation of the free radicals,including thechem-ically reactive products of oxygen metabolism called reactive oxygenspecies(ROS)31.Under physiological conditions,these metabolitesare involved in the signaling events r
38、egulating different cellular pro-cesses such as differentiation,proliferation,autophagy and survival32.However,if ROS production is beyond the capacity of the cellularantioxidant system,it may lead to the irreversible oxidative stressand cell death 33.The physiological ROS level is maintained bythe
39、scavenging molecules such as glutathione,dismutase,peroxidase,thioredoxin,catalase,and superoxide 33.In addition,adverse effectof ROS can be also minimized by an adequate DDR response 34.Ahighly efficient ROS scavenging system or generally lower ROS levelsin CSC may contribute to the high resistance
40、 of CSC populations togenotoxicstressdescribed for differenttumorentities 3538(Table 1).Besides the above-described intrinsic mechanisms underlying CSCradioresistance,the CSC fate under physiological conditions and duringtreatment is tightly regulated by a broad range of extrinsic microenvi-ronmenta
41、l stimuli 39,40.Within a tumor,CSCs can reside in specificniches occupying different locations including hypoxic,perivascularandinvasivetumorareasthatcandynamicallychangeduringtumorde-velopmentand treatment41,42.A CSC niche is defined byvarious sol-uble factors,extracellular matrix(ECM)elements and
42、direct cellcellinteractions via cell surface molecules.Depending on oxygen tension,a niche occupied by CSCs may define differentiation,self-renewal andtreatment resistance of the CSCs.The CSCs residing in the hypoxic64M.Krause et al./Advanced Drug Delivery Reviews 109(2017)6373Table 1The mechanisms
43、of CSC radioresistance.MechanismSignaling pathwayCSC populationInhibition of CSC resistanceCSC originReferenceActivation of DNA repairATR-Chk1PARPCSCs enriched under selective culture conditionCombined inhibition of ATM(KU55933)and PARP(olaparib),ATR(VE821)and Chk1(SCH900776 andCHIR-124)Primary pati
44、ent derivedglioblastoma cell lines19ATM-Chk1CSCs enriched under selective culture conditionInhibition of ATM with KU-55,933Primary patient derivedglioblastoma cell lines20Myc-dependent activation of Chk1 and Chk2PKH26+nasopharyngeal carcinoma cellsc-Myc siRNA-mediated knockdownCell lines17ATM-ZEB1-C
45、hk1EMT-mediated breast cancer cell reprogrammingZEB1 shRNA-mediated knockdownCell lines21ATR-Chk1ATM-Chk2CD133+glioblastoma cellsInhibition of Chk1 and Chk2 withdebromohymenialdisine(DBH)Glioma xenografts,clinicalsamples,cell lines16,18Chk2ALDH+prostate cancer cellsALDH inhibition with N,N-diethylam
46、inobenzaldehyde(DEAB)and galiellalactone,WNT pathway inhibitionwith XAV939 and CTNNB1 siRNA-mediatedknockdownCell lines22ATM-Chk2CD24/CD44+breast cancer cellsInhibition of ATM with KU55933Clinical samples,cell lines15Chk1CD133+CD44+prostate cancer cellsChk1 shRNA-mediated knockdownCell lines23PARPCD
47、133+glioblastoma cellsPARP inhibition with olaparibClinical samples24Upregulated DNA repair genes(BRCA1,Exo1,Rad51,Mre11a,Ku70,DNA PKC,Chk1,Fan1,Msh2)CD133+lung cancer cellsn/aCell lines14Protection from oxidative stressby ROS scavengingOverexpression of the genes controllingglutathione biosynthesis
48、(FoxO1,Gss,Gclm)CD24medCD49fhighmurine mammary epithelial cells;CD44+CD24/lowbreast cancer cells;Thy+CD24+mousemurine mammary tumor cells isolated from MMTV-Wnt1tumorsDecrease of glutathione withL-S,R-buthioninesulfoximine(BSO)which inhibits glutamate-cysteineligaseClinical samples,mouse tumors29Ove
49、rexpression of the genes controlling ROSdetoxification(MT3,Prdx3,Apoe,Prdx4,Nme5,Gpx1,Bnip3)CD24/lowbreast cancer cellsEctopic expression of CD24Cell lines28Activation of anti-apoptoticpathwaysWNT/-cateninPI3K/mTORCD44v6-mediated activation of WNT/-catenin andPI3K/mTOR signaling pathwaysCD44v6 siRNA
50、-mediated knockdownCell lines43WNT/-cateninALDH+prostate cancer cellsALDH inhibition with N,N-diethylaminobenzaldehyde(DEAB)and galiellalactone,WNT pathway inhibitionwith XAV939 and CTNNB1 siRNA-mediatedknockdownCell lines22WNT/-cateninEnrichment of CSC by in vivo irradiation using orthotopicglioma
