dna damage repair pathways in cancer stem cells

Review

DNA Damage Repair Pathways in Cancer Stem Cells

Marcello Maugeri-Sacca1, Monica Bartucci1, and Ruggero De Maria2

AbstractThe discovery of tumor-initiating cells endowed with stem-like features has added a further level of

complexity to the pathobiology of neoplastic diseases. In the attempt of dissecting the functional properties

of this uncommon cellular subpopulation, investigators are taking full advantage of a body of knowledge

about adult stem cells, as the "cancer stem cell model" implies that tissue-resident stem cells are the target

of the oncogenic process. It is emerging that a plethora of molecular mechanisms protect cancer stem cells

(CSC) against chemotherapy- and radiotherapy-induced death stimuli. The ability of CSCs to survive

stressful conditions is correlated, among others, with a multifaceted protection of genome integrity by a

prompt activation of the DNA damage sensor and repair machinery. Nevertheless, many molecular-

targeted agents directed against DNA repair effectors are in late preclinical or clinical development while

the identification of predictive biomarkers of response coupled with the validation of robust assays for

assessing biomarkers is paving the way for biology-driven clinical trials. Mol Cancer Ther; 11(8); 162736.

2012 AACR.

IntroductionMammalian cells have to constantly face genotoxic

injuries due to exposure to endogenous and exogenousagents. Biochemical reactions generate reactive oxygenspecies (ROS) that avidly bind to nucleic acids. In addi-tion, DNA chemical bonds physiologically undergo spon-taneous degradation. DNA is also attacked by a varietyof environmental mutagens of chemical, physical, andbiologic origins (1). Given the heterogeneity of DNA-damaging agents and the correlated diversity of geneticlesions, eukaryotic cells exploit distinct, albeit partly over-lapping, repair mechanisms (Table 1). In this manner,each repair avenue is engaged according to the type oflesion, even though the repair activity is carried outthrough a sequential recruitment of sensors, transducers,mediators, and effectors. The biologic outcome of DNAdamage depends on several factors. For instance, a dis-tinct response pattern characterizes different cell types, asshown by an extraordinary ability of adult stem cells torepair the genetic code comparedwith their offsprings (2).Additional determinants of cell fate are the extent of thelesion and the rapidity of its repair. Therefore, cell fate is

decided on the basis of the balance between tissue needsand damage severity. This is a process that culminates in acellular response that can span from transient cell-cyclearrest to senescence induction or apoptosis.DNA repair pathways encompass the nucleotide exci-

sion repair (NER), base excision repair (BER), mismatchrepair (MMR), direct repair, and the double-strand break(DSB) recombinational repair. The NER pathway correctsbulky helix-distorting lesions caused by chemicals andionizing radiations, theBER system targets small chemicalalterations (base modifications), whereas MMR removesnucleotidesmispaired arising from replication errors. Thesimplest repair mechanism is the monoenzymatic directrepair, a one-step methyl transfer reaction operated bythe O6-methylguanine methyltransferase (MGMT) thatdefends cells against alkylating agentgenerated lesions.The more challenging DSB repair is characterized by adifferent degree of fidelity in relation to the phase of thecell cycle.While the error-free homologous recombinationrepair (HRR) dominates in dividing cells, the G1 phaseactingnonhomologous end-joining (NHEJ) is error-prone,as a template for recombination is unavailable. These 2pathways repair themajority of chemotherapy- and radio-therapy-induced damage. Finally, cell-cycle checkpointcomponents, suchas ataxia telangiectasiamutated (ATM),ataxia telangiectasia/Rad3-related kinase (ATR), andcheckpoint kinases (Chk1 and Chk2), become engagedunder replication stress or consequently to DSBs. Thesecell-cycle arrest mechanisms allow the recruitment ofeither DNA repair effectors or, when a cell is irreversiblydamaged and the repair fails, proapoptotic molecules (3).While these mechanisms preserve genome integrity by

preventing transforming mutations, cancer cells improp-erly activate DNA repair pathways to overcome manystandard anticancer treatments. This has fostered the

Authors' Afliations: 1Department of Hematology Oncology and Molec-ular Medicine, Istituto Superiore di Sanita; and 2Regina Elena NationalCancer Institute, Rome, Italy

Note:M. Maugeri-Sacca and M. Bartucci contributed equally to this work.

Corresponding Authors: Marcello Maugeri-Sacca, Istituto Superiore diSanita, Viale Regina Elena 299, Rome 00161, Italy. Phone: 0039-0649904451; Fax: 0039-0649387087; E-mail:[email protected]; and Ruggero De Maria, Regina ElenaNational Cancer Institute, Via E. Chianesi, Roma 53,00144, Italy. Phone:0039-0652662726; Fax: 0039-0652665523; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-11-1040

2012 American Association for Cancer Research.

MolecularCancer

Therapeutics

www.aacrjournals.org 1627

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Published OnlineFirst July 25, 2012; DOI: 10.1158/1535-7163.MCT-11-1040

development of DNA damage pathwayinterferingagents, and many of these compounds are undergoingclinical trials. In such a scenario, the functional character-ization of cancer stemlike cells endowed with multipleprotective mechanisms (4), even including an extremeproficient DNA repair machinery, is crucial for sharpen-ing the therapeutic potential of these compounds.

DNA Repair in Adult Stem CellsAdult stem cells are a dedicated pool of undifferenti-

ated cells that maintain tissue homeostasis by counter-acting cell loss due to physiologic cellular turnover ortissue injuries. To do this, stem cells have the uniquecapacity to self-renew through which they give rise, at

each division, to a daughter cell that retains the parentalphenotype to avoid depleting the original pool and asecond cell that differentiates to finally acquire tissue-specific functions. Because stem cells ensure the lifetimefunction of tissues, they are equipped with multipleprotective mechanisms for constraining harmful insults.The hematopoietic system represents the benchmark

for dissecting DNA-protectingmechanismswithin a hier-archical context. After irradiation, the hematopoieticcompartment responds in a different way, with adulthematopoietic stem cells (HSC) displaying higher radio-resistance than their progeny (5). This differential patternof response preserves tissue homeostasis, thus enablingHSCs to fulfill local and systemic needs. To accomplish

Table 1. DNA repair pathways, target lesions, and diseases associated with DNA repair defects

DNA repairpathways Pathway effectors Target lesions/functions

Human disease associat-ed with DNA repairdefects

NER XPA, XPB, XPC-RAD23B,XPD, XPE, XPF, XPG,RPA, TFIIH, ERCC1

Bulky and helix-distortinglesions

Xeroderma pigmentosum,Cockayne syndrome,trichothiodystrophy

BER APE1, MUTYH, UNG,OGG1, NEIL1, NEIL2,NEIL3, NTHL1, MPG,TDG, SMUG1, POLb,XRCC1, APTX, TDP1,PNKP, LIG1, LIG3, FEN1,PCNA

Base modications Cancer predisposition,neurodegenerativedisorders,immunodeciency

MMR MSH2, MSH3, MSH6,MLH1, PMS2, EXO1

Sequence mismatches Lynch syndrome(hereditary nonpolyposiscolorectal cancer), Lynchsyndrome variants,sporadic colon cancer,noncolonic tumors

MGMT pathway MGMT (monoenzymaticpathway)

Alkylation (includingmethylation) at the O6position of guanine

HRR BRCA1, BRCA2, RAD50,RAD51, MRN, XRCC2,XRCC3

DSBs- SG2 phase acting Hereditary breast ovariancancer syndrome

NHEJ KU70, KU80, XRCC4,DNA-PKc, DNA ligase IV

DSBs- G1S phase acting Syndromes with braindevelopment defectsand immunologicabnormalities

Fanconi anemiapathway

FANCA, FANCC, FANCD1/BRCA2, FANCD2,FANCE,FANCF, FANCG,FANCI, FANCJ, FANCL,FANCM, FANCN

Interstrand DNA cross links Fanconi anemia

ATM/ATR-mediatedsignaling

ATM, ATR, CHK1, CHK2,MRN, ATRIP, MDC1,53BP1, MCPH1/BRIT1,RNF8, RNF168/RIDDLIN,RAD17, RAD9RAD1HUS1 (9-1-1) complex

Cell-cycle checkpoints Ataxia-telangiectasia,Nijmegen breakagesyndrome

Maugeri-Sacca et al.

Mol Cancer Ther; 11(8) August 2012 Molecular Cancer Therapeutics1628



this function, HSCs are maintained in a quiescent statewithinprivileged bonemarrowmicroenvironments, com-monly referred to as niches, which protect HSCs fromexhausting their replication potential andminimizing theexposure to DNA-damaging metabolic products (6). If onthe one hand, quiescence ensures longevity to HSCs; onthe other hand, the slow replication kinetics forces them,when required, to adopt the low-fidelityNHEJ. Therefore,this short-term survival strategy is theoretically burdenedby potential detrimental effects on genome integrity andcould account for post-chemotherapy/radiotherapyhematologic malignancies and age-related blood disor-ders. It is worth noting that when forced to cycle, HSCscontinue to opt for DNA repair instead of cell death (7).Unlike quiescent HSCs, however, cycling hematopoieticprecursors take advantage of the error-free HRRs. Thisobservation further highlights the crucial role of cell-cyclekinetics on the fidelity of DNA repair. Finally, HSCs atdifferent ontogenetic stages express a distinct pattern ofresponse uponDNAdamage. Highly proliferative umbil-ical cord bloodderived HSCs display a slower rate ofDSBs compared with more differentiated progenitorstogether with a massive recruitment of apoptotic media-tors (8). This indicates that programmed cell death is themain outcome of DNA damage at this developmentalstage. This response is consistent with the need to estab-lish a proficient pool maintaining blood homeostasis forthe whole lifespan.Similarly, epidermal stem cells express greater resis-

tance to DNA-damaging agents than to all other cells ofthe epidermis, as indicated by higher expression of anti-apoptotic molecules, shorter p53 activation, and enhancedNHEJ activity (2). These mechanisms are shared by othertissue-resident stem cells, thus indicating that stem cellshave evolved highly efficient repair mechanisms (2). Not-withstanding, quantitative (reduced self-renewal or differ-entiation potential) or qualitative (mutations) alterationscan perturb stem cell homeostasis, leading to the onset ofdegenerative and neoplastic diseases, respectively.

The Cancer Stem Cell ModelThe "cancer stem cell theory" has captured great atten-

tion following the identification of a rare population ofleukemia-initiating cells possessing stem-like features (9)and has been further strengthened by the isolation andcharacterization of tumor-initiating, stem-like cells inalmost all solid tumors (1013). According to this model,an uncommon population of tumor cells endowed withself-renewal ability and therefore referred to as cancerstem cells (CSC), accounts for tumor initiation, progres-sion, and treatment failure. This allowed to envisiontumors as organized, like normal tissues, in a hierarchicalmanner (hierarchical model) with a CSC occupying theapex of the pyramid and serving as the precursor ofwholetumor population. The logic behind the CSC theory orig-inally fueled an intense debate having questioned the"clonal evolution model." This theory postulates that

mutant clones, each one with the same ability to prolif-erate and to retain tumorigenicity, cohabit the tumorcompetingwith each other to ensure nutrients and endureto microenvironmental perturbations. However, compel-ling evidence indicates that only few cancer cells areactually tumorigenic and that these tumorigenic cellscould be considered CSCs. To this regard, compared withtotal bulk cells, CSCs express higher levels of stem cellgenes (14, 15), possess clonogenicity in vitro, and highertumorigenic potential in vivo (10, 16). Moreover, theunexpected complexity of the tumor hierarchy has beenrecently confirmed following the isolation of multiplecolon cancerinitiating clones with distinct propertiesupon serial transplantation into the murine background(Fig. 1; ref. 17).The conclusion drawn by the stem-like phenotype of

tumor-initiating cells is that stem cells are the target of theoncogenic process. This has fostered the translation ofknowledge about stem cell biology to the pathobiology ofcancer, based on the assumption that stem cells under-goingmalignant transformation generate cancer cells thatretain, although in a distorted manner, their functionalproperties. The concept that CSCs are reminiscent of theirorigin is supported by the imbalanced distribution of self-renewal effectors between CSCs and their differentiatedprogeny (18). This is corroborated by the preferentialdepletion of CSCs following the pharmacologic abro-gation of self-renewal pathway components (19). It is alsobelieved, although not fully proven yet, that the necessarystimuli exploited by CSCs to retain both self-renewal anddifferentiation ability are the results of their interactionwith the environment in which they reside (20). Consis-tent with this, paracrine-acting, self-renewalassociatedpathways have been linked with the epithelialmesen-chymal transition (21), a genetic program inducing bothprometastatic traits and stem-like features in cancer cells(22). The gain of "stemness" by neoplastic cells is alsoelicited by physiologic conditions existing within niches,such as hypoxia and low pH (23, 24). In turn, CSCs thrivealso by their ability to recreate optimalmicroenvironmen-tal conditions, as suggested by their direct differentiationinto endothelial-like cells (25, 26).The relative abundance of CSCs in tumoral tissues has

been correlated with both the prognosis of patients withcancer and the efficacy of anticancer treatments. Aninverse relationship exists between the in vitro growthpotential of CSCs and clinical outcomes of patients withglioblastoma treated with standard surgical resectionfollowed by adjuvant chemoradiotherapy (27). Similar-ly, an increased sphere-forming ability has beenreported following neoadjuvant chemotherapy fortreating patients with breast cancer (28). This findingsuggests greater chemoresistance of CSCs in compari-son to the bulk of tumor cells. An optimal discoveryvalidation path of CSC-related signatures might havedeep implications for both individual risk assessmentand identification of targetable pathways. For instance,an "invasiveness gene signature" composed of 186

DNA Damage Repair and Cancer Stem Cells

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differentially expressed genes in breast CSCs comparedwith normal breast epithelium has been associated withoverall and metastasis-free survival of patients withbreast cancer (29). More recently, germ line polymorph-isms in colon CSCrelated genes have been linked to thetime to tumor recurrence in high-risk patients withstage II and stage III colon cancer treated with standardadjuvant chemotherapy (30). However, the relativesmall cohort of patients examined in these studies andtheir retrospective nature impose further investigationsto assess the prognostic/predictive value of CSC-relat-ed parameters in the clinical setting.

DNA Repair in CSCsGiven the homologies existing between stem cells and

their malignant counterpart, it is not surprising that CSCspossess similar defensive mechanisms. The connectionbetween DNA repair signals and CSCs chemoresistancestems from studies carried out in high-grade primarybrain tumors. In a pioneering report, CD133 glioblasto-ma stem cells activated ATM and Chk1 more promptlythan the CD133 counterpart (31). This molecularresponse enabled CD133 cells to survive ionizing radi-ation, as opposed to the CD133 population that under-went cell death. Notably, radiosensitivity was restored bythe pharmacologic abrogation of Chk1 and Chk2. Subse-quent evidence, however, failed to confirm that the glio-blastoma stem cells pool reacts with enhanced DNArepair activity following exposure to ionizing radiation.In particular, radioresistance properties were linked tocell-cycle kinetics, as indicated by the significant increasein the population doubling time and enhanced basalactivation of Chk1 and Chk2 (32). This elongated cellcycle, therefore, theoretically provides more time for

repairing DNA damage. To further intricate this picture,a direct comparison of radiosensitivity between glioblas-toma stem cells and apanel of established glioma cell linesrevealed that CD133 cells exhibit reduced DSB repairability (33). Cell-cycle analysis revealed that althoughglioblastoma stem cells possessed intact G2 checkpoint,they displayed deficient activation of the intra-S-phasecheckpoint. Because the latter checkpoint is crucial formaintaining genome integrity, chemotherapy could par-adoxically lead to the emersion of genetically unstableCSCs, thus explaining the pattern of disease progressionduring sequential chemotherapeutic regimens. However,preclinical studies continue to be inconsistent. Recently,unsupervised hierarchical clustering analysis of geneexpression data, provided by The Cancer Genome AtlasNetwork, revealed that high-grade primary brain tumorscan be grouped in subtypes (proneural, classical, mesen-chymal, andneural). Eachmolecular asset is characterizedby a different composition of somatic mutations inmasteroncogenes and oncosuppressors such as EGF receptor(EGFR), CDKN2A, PDGFRA, NF1, p53, and PTEN (34).Therefore, this genetic heterogeneity could mirror a dif-ferent DNA damage repair proficiency among subtypes,thus providing a possible explanation for the conflictingresults discussed above.Next, theMGMTpromotermeth-ylation status is routinely assessed in patients diagnosedwith glioblastomamultiforme. It is known that theMGMTpathway is adopted by glioblastoma cells to overcometemozolomide cytotoxicity and, to a similar extent, thisenzyme protects glioblastoma stem cells from alkylatingagents (35).Notwithstanding, a comparative evaluationofthe MGMT promoter methylation pattern between surgi-cal samples and paired glioblastoma-derived neuro-spheres indicated that epigenetic silencing of MGMT isenriched in putative glioblastoma stem cells (36), thus

Figure 1. The evolution of theCSC concept. A, the CSCmodel originally postulated thata stem-like cell occupying theapex of the tumor pyramid is theprecursor of the whole tumorpopulation, and its intrinsicplasticity accounts for theheterogeneity characterizingcancer tissues. B, the apex ofthe pyramid seems to be morecomplex, being composed bydistinct CSCs, with a distinctgenetic background and adifferent biologic behavior.TAC, transit-amplifying cells;TDC, terminally differentiatedcells.





sheddingdoubts on the biologic relevance of this pathwayon survival of temozolomide-treated glioblastoma stemcells. High-grade primary brain tumors are also known toaberrantly activate the phosphoinositide 3-kinase (PI3K)/Akt pathway (37), an oncogenic axis functionally inter-connectedwith theDNArepairmachinery, as highlightedby the ability of PI3K or Akt inhibitors to hamper theremoval of radiation-induced DNA damage (38). It isworth considering that the pharmacologic abrogationof Akt impaired glioblastoma stem cells fitness andabrogated neurosphere formation (39), thus allowing topostulate that Akt inhibitors could be exploited as che-motherapy-enhancing agents. Although the mechanisticlink between checkpoint-associated molecules and glio-blastoma stem cells survival upon genotoxic injuries isstill debated, similar survivalmechanisms are adopted byother CSC types. For instance, significant increases in theexpression of DNA repair- and cell-cyclerelated geneshave been observed in pancreatic CSCs compared withbulk cells after challenge with gemcitabine (40). UsingOncomine database coupled with Ingenuity PathwaysAnalysis (IPA), a significant increase in DNA copy num-ber of BRCA1 and RAD51 has been observed in prostaticCSCs compared with adherent population isolated fromthe primary site (41). Moreover, both colon and lungCSCs, unlike their differentiated progeny, efficiently acti-vate Chk1 when exposed to standard chemotherapeuticagents (42, 43). While the aberrant activation of G2Mcheckpoint controllers conferred chemoresistance, theirpharmacologic inhibition significantly increased chemo-sensitivity by triggering amodality of cell death, knownasmitotic catastrophe, aimed at eliminating mitosis-incom-petent cells. These data indicate that a proficient DNArepair mechanism exploited by CSCs may be responsiblefor the partial inefficiency of current treatments and urgethe need for a well-designed CSC-tailored therapy.Whether brain tumor models have been widely used

for studying DNA repair pathways at the preclinicallevel, breast cancers harboring germ line mutations in theHRR-associated proteins BRCA1 and BRCA2 are thebenchmark for proof-of-principle clinical trials aimed atassessing the antitumor activity of DNA repair pathwayinhibitors. Enhanced DNA repair ability has been alsodescribed in breast CSCs whose isolation and character-ization provided the first hint supporting the CSC modelin solid tumors (10). Transcriptional profiling of the puta-tive CSC population isolated from themammary gland ofp53-null mice indicated that these cells were enriched inboth DNA repair- and self-renewallinked genes (44).Array-based gene expression analysis conducted by theAffymetrix HuGene 1.0 ST Array, conducted in our lab-oratory, revealed that breast CSCs, isolated from primaryxenograft-derived tumors, and metastatic derivativesexhibit higher levels of DNA repairlinked effectors suchas BRCA1, ATR, ATM, and Chk1 when compared withdifferentiated tumor cells (Bartucci and colleagues,unpublished results). Furthermore, mammospheres fromthe commercial cell line MCF-7 displayed a more active

DNA single-strand break repair (SSBR) pathway in com-parison to the bulk population, as indicated by higherlevels of the SSBR-associated protein APE1 (45). More-over, long-term exposure of MCF-7/ADR cells to doxo-rubicin led to gaining stem-like properties coupled withenhanced chemoresistance-conferring mechanisms (46),as documented by the increased expression of genesencoding multidrug resistancerelated proteins and thecyclophosphamide-metabolizing enzymealdehydedehy-drogenase 1. Radioresistance of breast CSCs seems to bealso sustained by lower concentrations of ROS (47). Thisphenomenon is due to an increased expression of freeradical scavenger systems, such as those belonging to theglutathione metabolism, which counteracts the effects ofwater radiolysis, themainmodality of ionizing radiationinduced cell death. This radioresistant phenotype wasreverted by the inhibition of glutathione metabolism,which restored breast CSC radiosensitivity and decreasedtheir clonogenic potential. To further enforce the inter-connection between stemness-associated pathways andDNA repair signals, it has been reported that the aberrantactivation of both the canonical WNT pathway and Aktconferred radioresistance to breast CSCs, whereas Aktneutralization sensitized breast CSCs to radiotherapy viathe inhibition of b-catenin (48). Although the role of DNArepair pathways in determining chemoradioresistance ofbreast CSCs seems to be less contradictory than in braintumors, data should be interpreted with caution. In fact,breast cancer is a constellation of molecular and clinicalentities, each one characterized by a distinct clinicalbehavior, a different molecular portrait, and a differentdegree of responsiveness to chemotherapy, hormone ther-apy, andmolecular-targeted agents. Therefore, themolec-ular taxonomy of breast cancer implies that optimaldevelopment of DNA repair inhibitors should be carriedout within well-defined genetic contexts.It is known that stem cell longevity is ensured by

prolonged exit from the cell cycle, a mechanism thatprevents the exhaustion of the replicative potential andlimitsDNAdamage (49). Experimental evidence indicatesthat both in vitro and in vivo, a subpopulation of slow-cycling tumor cells is mostly spared by chemotherapy-induced death when compared with the bulk of the cells(50, 51). Ovarian and pancreatic cancer label-retainingpopulation, both encompassing the operative criteria tobe defined as CSCs, were able to survive, unlike non-labelretaining cells, standard chemotherapeutic agents(50, 51). The existence of quiescent CSCs has fostered thedevelopment of pharmacologic strategies able to target"dormant" cells, and some compounds including cyto-kines (IFN-a and granulocyte colony-stimulating factor)or chemicals (arsenic trioxide) seem to be endowedwith such properties (52). The list of potential "quies-cence-disrupting" agents has been further implementedwith epigenetic-acting histone deacetylase inhibitors(HDACi). The first-in-class HDACi vorinostat, a com-pound approved for treating refractory cutaneous T-celllymphoma, successfully induced apoptosis in quiescent





chronic myelogenous leukemia stem cells when com-bined with imatinib mesylate (53). However, given thetight relationship existing between DNA repair strategiesand cell cycle, CSCs are probably forced to use the error-prone NHEJ (Fig. 2). Therefore, quiescence potentiallycontributes to both chemoresistance and genetic instabil-ity of CSCs, whereas co-targeting quiescence-associatedmolecules and DNA repair pathway effectors might con-tribute to an efficient eradication of CSCs.

DNA Repair Pathway Inhibitors and CompanionBiomarkersEarly clinical trials aimed at assessing the activity of

DNA repair inhibitors have been carried out with theMGMT-depleting agents O6-benzylguanine and lome-guatrib in combination with carmustine and temozo-lomide, respectively (54, 55). Although an efficientdepletion of the target was documented in the phar-macodynamic assay, results were disappointing; per-haps, the unacceptable toxicity observed with standardschedules was required to adopt suboptimal chemo-therapy doses.More recently, a second wave of clinical trials with

chemotherapy-enhancing therapeutic approaches hasbeen conducted for determining the antitumor activityof PARP inhibitors. Currently, at least 9 of these mole-cules are in clinical or late preclinical development.PARPs are nuclear enzymes with multiple functionsand, among components of the family, PARP-1 andPARP-2 are involved in single-strand repair via theBER pathway. The molecular background underlyingthe development of PARP inhibitors is a modality of

genegene interaction known as synthetic lethality.According to this model, while a mutation confers anadvantage for cancer cells, the concomitant pharmaco-logic abrogation of a redundant pathway significantlyaffects cell fitness. Because BRCA-deficient cells haveimpaired HRRs, they are forced to use the BER pathwayfor repairing persistent SSBs. As a result, PARP neu-tralization makes BRCA-deficient cells unable to usethis alternative pathway when exposed to DNA-dam-aging agents, thus leading to cell death. This approachhas been exploited for targeting breast and ovariancancers carrying BRCA1 or BRCA2 germ line mutations.A phase II multicenter study conducted in patients withadvanced, refractory breast cancer whose tumors har-bored BRCA mutations evaluated 2 different doses ofolaparib (AZD2281) in 2 sequential cohorts (27 27patients; ref. 56). The overall response rate was 41%(11 patients) and 22% (6 patients) with olaparib given at400 mg and 100 mg, respectively, with an acceptablesafety profile. Such promising, although initial, resultswere confirmed in 57 BRCA-mutated carriers with ovar-ian cancer (57). More recently, a phase II, open-label,nonrandomized study conducted in patients withhigh-grade serous and/or undifferentiated ovarian can-cer and advanced triple-negative breast cancer con-firmed positive results against ovarian cancer carryingBRCA1 or BRCA2 mutations. Although to a lowerextent, the antitumor efficacy was also documented inthe nonmutant background (58). However, no con-firmed objective responses were reported in patientswith breast cancer. Iniparib (BSI-201), another PARPinhibitor, has been recently evaluated in a randomized,open-label, phase II study trial in combination with

Figure 2. The relationshipbetween cell cycle and DNArepair delity. When damaged,quiescent/slowly cycling CSCsare forced to use the error-prone NHEJ pathway. Thisrepair strategy couldparadoxically generate a newmutation that is passed down tothe progeny. The resultingoffspring can therefore displayenhanced malignant propertiessuch as metastatic ability orincreased chemoresistance.





carboplatin/gemcitabine for treating metastatic triple-negative breast cancers (59). Consistent with the factthat this breast cancer subtype displays dysregulation ofBRCA1 and, therefore, shares molecular and clinicalfeatures with hereditary BRCA1-related breast cancers(BRCAness phenotype; ref. 60), adding iniparib toalkylating agentbased chemotherapy significantlyimproved both clinical benefit and overall response rate.The clinical benefit rate was 56% in the experimentalarm compared with 34% in the chemotherapy-alonearm. The overall response rate also favored the inves-tigational treatment (32% vs. 52%). All other endpoints(median progression-free survival and median overallsurvival) were improved with the iniparib-containingregimen, although the trial was not powered to lookat long-term outcomes. However, the subsequent phaseIII trial unexpectedly failed to improve overall andprogression-free survival, and molecular analysis isunderway in the attempt of identifying the subset ofresponder patients (61). A possible explanation for suchunsatisfying results is that iniparib, as opposed to othersPARP inhibitors, mainly acts by inducing cell-cyclearrest in G2M phase rather than inhibiting PARP-1and PARP-2, at least at physiologic drug concentrations.While current molecular-targeted agents are directed

against proto-oncogenic proteins, lethal interactionbased therapy is offering the opportunity for targeting,although indirectly, deregulated oncosuppressors. Thisrationale has been also proposed for developing Chk1inhibitors. Because p53-defective cells are unable toundergo G1 arrest, they depend on alternative checkpointactivators to arrest the cell cycle in response to DNAdamages. Conversely, cells with intact p53-dependentcheckpoint are expected to be unperturbed, thus implyingthat normal cells should be spared from the sensitizationto DNA-damaging agents. Many Chk1 inhibitors showedchemosensitizing properties in the preclinical setting andare undergoing early phases of clinical development(AZD7762, PF-477736, SCH900776, LY2606368; ref. 3).Notwithstanding, 2 main concerns recently arose frompreclinical evidence and early clinical data. The prefer-ential antitumor activity of Chk1 inhibitors against p53-defective cells has been questioned. In particular, bothshort-term cell survival and long-term colony-formingability have been reported to be independent from p53status following Chk1 neutralization (62). This findingbrings into question the predictive value of p53 status and,therefore, could negatively impact the biomarker-drivendevelopment of Chk1 inhibitors. Second, although Chk1antagonists were thought to have a favorable therapeuticindex, two phase I dose-escalation trials with AZD7762in combination with either gemcitabine or irinotecanreported an unexpected cardiotoxicity, which led to with-drawing this compound from themarket (63, 64). Becausecardiac dose-limiting toxicity was also observed with theChk1 inhibitor SCH900776 in combination with gemcita-bine (65), and considering that the above-mentioned che-motherapeutic agents are not associatedwith an increased

risk of cardiac events, whether cardiotoxicity is a classeffect of Chk1 antagonists needs to be urgentlyaddressed. Agents targeting G2 phase checkpoint mem-bers acting downstream of Chk1, such as Wee1, havebeen developed with the same conceptual approachproposed for Chk1 inhibitors. The Wee1 inhibitorMK-1775 potentiates the activity of many DNA-damag-ing agents such as platinum derivatives, both in vitroand in vivo, mainly in a p53-dependent manner (66).Although MK-1775 enhanced radiosensitivity in com-mercial glioblastoma cell lines without affecting normalhuman astrocytes, similar properties have not beenconfirmed against glioblastoma stem cells (67).Such an expanding pipeline of DNA damageinter-

fering agents has fostered the identification of pharma-codynamic biomarkers coupled with robust and reliabletests for detecting DNA damagerelated endpoints.DSB-induced g-H2AX foci are widely used as a biodo-simeter for measuring the extent of genotoxic injuriesinduced by the exposure to chemicals or radiation. Inrecent years, this parameter has moved from the labo-ratory to be used in early clinical trials with DNAdamage repair antagonists (68). One of the most chal-lenging aspects in the early development of molecular-targeted agents is the need for multiple biologic samplesfor monitoring the target over time. To overcome thisdrawback, g-H2AX levels have been evaluated in healthtissues (circulating blood cells, buccal cells, hairs) as asurrogate parameter of drug-induced DNA damage.However, the exploitation of surrogate tissues insteadof the tumor for measuring biologic outcomes has someintrinsic limitations, correlated with both the cell typeevaluated and its replicative state. To this end, thedetermination of g-H2AX on circulating tumor cellswill enable investigators to conduct multiple measure-ments in the target cells in a minimally invasive way(69). Finally, a high-throughput screening system(RABIT-Rapid Automated Biodosimetry Tool), basedon an established g-H2AX immunofluorescence assay,has been developed to screen thousands of samplesper day (70). When considering DNA repairlinkedbiomarkers, however, the attention turns to the enzymerepair cross-complementation group 1 (ERCC1), anNER component whose levels have been traditionallyassociated with the benefit of patients with nonsmallcell lung cancer (NSCLC) platinum-containing doub-lets. Although the predictive value of this biomarkerremains to be addressed, a recent meta-analysis sug-gested that low ERCC1 levels are associated with ahigher objective response and longer survival inpatients with advanced NSCLCs (71). In addition, themolecular analysis of 1,207 NSCLCs revealed a strongassociation between low ERCC1 mRNA levels andEGFR-activating mutations (72). This observationimplies that the co-detection of such molecular deter-minants could identify a subset of NSCLCs with amarked sensitivity to both EGFR-tyrosine kinaseinhibitors and platinum-containing chemotherapy. The





connection between DNA repair ability and outcomes ofpatients with NSCLCs receiving platinum-based che-motherapy has been further enforced by the existence ofan inverse relationship between DNA repair capacity,measured in vitro in lymphocytes with the host cellreactivation assay, and patients survival (73). Finally,it is known that approximately 15% of sporadic colo-rectal cancers are characterized by microsatellite insta-bility (MSI; ref. 74). This form of genetic instability issustained by alterations in MMR components, consist-ing of either germ line mutations in one of the MMRgenes (MLH1, MSH2, MSH6, and PMS2) or epigeneticsilencing of MLH1. This colorectal cancer subtype pre-sents distinct clinicopathologic features such as right-sided location, lymphocytic infiltration, mucinous his-tology, and poor differentiation. TheMSI phenotype hasbeen associated with better prognosis and reducedlikelihood of metastasis compared with microsatellite-stable tumors, even though patients whose tumors har-bor the MSI phenotype have a less pronounced benefitfrom 5-fluorouracilbased chemotherapy

ConclusionsThe exact definition of the target population is a

crucial element for optimal preclinical development ofmolecular-targeted agents. Growing evidence points toCSC eradication as the most valuable strategy forachieving long-lasting tumor remission. However,CSCs are protected against standard medical treatmentsby multiple mechanisms, also including the abnormalactivation of DNA damage repair signals. We believethat 3 main questions need to be addressed to fullydissect DNA repair pathways as therapeutic targetswithin the pyramidal organization of tumors. First,commercial cancer cell lines have been traditionallyused for generating tumors in mice, although these cellsare unable to give rise to a tumor resembling the humandisease. Given the possibility to expand in vitro CSCs,the generation of CSC-based tumor xenografts able torecapitulate the parental tumor into the murine back-ground is now considered as the gold standard forevaluating the anticancer properties of experimentalagents. This opportunity has been welcomed as a majoradvance in experimental oncology. The genetic hetero-geneity existing at the apex of the tumor pyramidshould be however considered. The distinct biologicbehavior of tumor-initiating subpopulations is indeedthe potential source of discrepant results, even withinthe same experimental model, coming from studiesaimed at dissecting the role of DNA damage pathwaysin CSC chemoradioresistance. Second, a rationale devel-opment of DNA repair inhibitors and, more in general,of molecular-targeted agents should be ideally carriedout within a defined genetic background. This stemsfrom evidence showing that the majority of biomole-cules used in the clinical setting are effective in thepresence of specific genetic alterations. When consider-

ing DNA repair inhibitors, 2 different, but complemen-tary, approaches could be exploited by taking advan-tage of high-throughput RNA interference screeningassays. These tools make it possible to identify molec-ular networks whose abrogation produces the expectedoutcome only in the presence of a predefined molecularparameter (mutation) and therefore to determine theextent to which DNA repair components are repre-sented in such circuits. Similarly, mutant effectors ofthe DNA repair machinery could be adopted as thereference parameter, thus allowing the identification ofpharmacologic strategies for targeting cells carryinggenetic defects in DNA damage pathways. The logicbehind this approach is the co-development of DNAdamage inhibitors and companion biomarkers, a pro-cedure that will enable physicians to plan clinical trialsin selected patient populations. It is plausible that thisapproach will significantly shorten the developmentalpath of new agents that can be evaluated, for instance, inmultiarm trials with an adaptive randomization designwhile avoiding the need for large randomized phase IIItrials in unselected patients. Third, chemotherapy-enhancing agents aimed at eliminating CSCs could beburdened by severe side effects due to "off-target"effects on normal stem cells. Although molecular-tar-geted agents have been traditionally considered to besafe, this paradigm is rapidly changing. Safety concernshave halted clinical trials with promising biomolecules.Neratinib, for instance, is an irreversible pan-ErbBreceptor tyrosine kinase inhibitor whose antitumoractivity was limited by diarrhea-related dose reduction(75). It is evident that a deeper characterization ofmechanisms protecting stem cells is needed, optimallyrequiring a panel of human stem cells representing themost critical tissues as "safety control" during preclin-ical development of potential anti-CSCs drugs. Webelieve that this is crucial to avoid clinical trials to stopearly and/or dose reduction of the chemotherapybackbone.

Disclosure of Potential Conicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: M. Maugeri-Sacca, M. Bartucci, R. De MariaWriting, review, and/or revision of themanuscript:M.Maugeri-Sacca,M.BartucciStudy supervision: R. De Maria

AcknowledgmentsThe authors thank Giuseppe Loreto and Tania Merlino for technical

assistance. Owing to constraints of space many excellent articles have notbeen cited for which we apologize.

Grant SupportThe study was supported by Italian Association for Cancer Research,

ItalianMinistry ofHealth, and ItalianMinistry forUniversity andResearch(R. De Maria).

Received January 3, 2012; revised March 28, 2012; accepted April 16,2012; published OnlineFirst July 25, 2012.





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2012;11:1627-1636. Published OnlineFirst July 25, 2012.Mol Cancer Ther

Marcello Maugeri-Sacc, Monica Bartucci and Ruggero De Maria

DNA Damage Repair Pathways in Cancer Stem Cells

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