Overcoming Multidrug Resistance in Cancer Stem Cells

Review ArticleOvercoming Multidrug Resistance in Cancer Stem Cells

Karobi Moitra

Department of Biology College of Arts and Sciences Trinity Washington University 125 Michigan Avenue NEWashington DC 20017 USA

Correspondence should be addressed to Karobi Moitra moitraktrinitydcedu

Received 9 August 2015 Revised 9 October 2015 Accepted 19 October 2015

Academic Editor Milica Pesic

Copyright copy 2015 Karobi MoitraThis is an open access article distributed under theCreative CommonsAttribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The principle mechanism of protection of stem cells is through the expression of ATP-binding cassette (ABC) transporters Thesetransporters serve as the guardians of the stem cell population in the body Unfortunately these very same ABC efflux pumpsafford protection to cancer stem cells in tumors shielding them from the adverse effects of chemotherapy A number of strategiesto circumvent the function of these transporters in cancer stem cells are currently under investigation These strategies includethe development of competitive and allosteric modulators nanoparticle mediated delivery of inhibitors targeted transcriptionalregulation of ABC transporters miRNAmediated inhibition and targeting of signaling pathways that modulate ABC transportersThe role of ABC transporters in cancer stem cells will be explored in this paper and strategies aimed at overcoming drug resistancecaused by these particular transporters will also be discussed

1 Introduction

Chemotherapy has long been the method of choice forthe treatment of metastatic tumors however cancer cellsfrequently develop an almost uncanny ability to resist theeffects of cancer chemotherapeutic agents This ability ofcancer cells to become simultaneously resistant to severalstructurally unrelated drugs that do not have a commonmechanism of action is known as multidrug resistance andcan severely impair the success of cancer chemotherapyCellular mechanisms of drug resistance arise in the cancercell itself due to either genetic or epigenetic alterations thatcan alter sensitivity of the drug In the clinical setting thesemay include pumping out of the drug by ABC transporters(ABCB1P-glycoprotein ABCC1 ABCG2 etc) sequesteringof drugs into vesicles and subsequent extrusion by exocytosisand reduced uptake of drugs such as water soluble drugs thatpiggyback on transporters and carriers that are used to bringnutrients into the cell Othermechanisms are the activation ofdetoxifying pathways such as the cytochrome P-450 pathwayand the cellular glutathione system and mechanisms thatrepair drug induced damage of cancer cells and disruptions inapoptotic signaling pathways allow cells to become resistant

to drug induced apoptotic cell death in cancer cells [1 2] Inpopulations of cancer cells exposed to chemotherapy morethan one of these mechanisms of multidrug resistance maybe present this phenomenon is known as multifactorial drugresistance

During treatment cells susceptible to chemotherapy arekilled and generally a few cells in the tumor remain andbecome resistant to drugs these resistant cells multiplyand the tumor eventually becomes unresponsive to treat-ment The unanticipated detection of cancer stem cells insolid tumors has drastically changed our outlook regardingcarcinogenesis and chemotherapy The implication of thisdiscovery is far-reaching and would for all intents andpurposes indicate that a self-renewing cancer stem cellpopulation is present in tumors More importantly thesecancer stem cell (CSC) populations unlike other cells areldquointrinsicallyrdquo resistant to chemotherapy This indicates thatsurviving cancer stem cells can propagate after chemotherapyand subsequently give rise to tumors [3]

In this review we will focus on the role of ABC (ATP-binding cassette transporters) in cancer stem cell (CSC)drug resistance and discuss strategies for overcoming ABCtransporter-mediated drug resistance in CSCs

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 635745 8 pageshttpdxdoiorg1011552015635745

2 BioMed Research International

2 The Cancer Stem Cell Model

The cancer stem cell (CSC) model expounds on the processby which established cancers are able to propagate [4] TheCSC theory proposes that a defined population of cancer cells(known as cancer stem cells) have the ldquoexclusiverdquo propertyto drive the growth and propagation of a tumor CSCs canalso give rise to progeny that have a limited ability to divide[5] However tumorigenic stem cells frequently lack some ofthe control mechanisms present in normal stem cells whereproliferation is tightly regulated and the genomic integrity ofthe cells is maintained If the doctrine that tumors containstem cells is assumed to be correct then we could possiblyinterpret the accumulation of mutations in these stem cells asthe basic ldquomultisteprdquo process of carcinogenesis The uncannyability of cancer stem cells to resist chemo- and radiotherapywould lead us to ask the question Does the ldquoinnaterdquo orldquointrinsicrdquo resistance of stem cells to radiation and toxinscontribute to the failure of some cancer therapies Andultimately askHow canwe exploit our knowledge of stem cellbiology to specifically target CSCrsquos in order to overcome drugresistance and improve therapeutic outcome in the clinic

3 ABC Transporters and Stem Cells

ATP-binding cassette transporters (ABC transporters) areintricate molecular pumps most of which catalyze the trans-port of awide array of substrates across biologicalmembranesagainst a concentration gradient by the hydrolysis of ATPThe human genome is known to encode 48 ABC transportergenes categorized into seven subfamilies ranging from Ato G [6] ABC transporters are classified based on thesequence and organization of their ATP-binding domain(s)that contain specific conserved motifs Walker A andWalkerB (present in all ATP-binding protein) however ABC trans-porters contain an additionalmotif the signaturemotif or theC-loop that is located upstreamofWalker Bmotif Functionaltransporters usually contain two transmembrane domains(TMDs) generally made up of 6ndash12 membrane-spanningalpha-helices that are primarily responsible for determiningsubstrate specificity Additionally there are two nucleotidebinding domains (NBDs) that bind and hydrolyze ATPproviding the energy for substrate translocation (Figure 1)ABC transporters may be expressed in stemprogenitor cellsderived from several types of normal tissue and also inhematopoietic cells Hematopoietic stem cells (HSCs) werefound to express high levels of ABCG2 andor ABCB1transporters [7] Mouse knockouts for ABCB1 ABCG2 orABCC1 revealed that mice are particularly sensitive to somecompounds such as mitoxantrone vinblastine ivermectinand topotecan indicating that these transporters may havea role in protecting the stem cells from toxic substances [8]Using the aid of Taqman low density arrays ABC transporterswere also found to be expressed in normal stem cells such ashematopoietic stem cells (HSCs) unrestricted somatic stemcells (USSCs) mesenchymal stem cells (MSCs) andmultipo-tent adult progenitor cells (MAPCs) [9] HSCs seemed to relyon a different repertoire of these transporters compared toother tissuecell types based on the fact that gene signatures

TMD 2

NBD 1 NBD 2

TMD 1

N

C

Figure 1 Schematic diagram of a typical ABC transporter(ABCB1P-glycoprotein) depicting the structural organization of afull transporter A full transporter typically contains 2 transmem-brane domains (TMDs) and 2 nucleotide binding domains (NBDs)

for ABC transporters were found to be radically differentbetween HSCs and other types of stem cells 16 transportersin total including ABCB1 and ABCG1 were discovered tobe consistently expressed at higher levels in HSCs whencompared to other transporters The transporters ABCA4ABCA8 ABCC9 and ABCG4 were consistently detected inMSCs and USSCs [9]

4 Distinguishing Characteristics andBiomarkers of CSCs

Cancer stem cells (CSCs) possess the unique property toevade radiotherapy and chemotherapy Compared to differ-entiated tumor cells CSCs have 4 distinguishing featuresthey are as follows (i) they are relatively quiescent (ii) theyhave a slow cycling rate (the relative quiescence and slowcycling rate afford these cells protection against chemother-apeutics that target rapidly dividing cells rather than a slowcycling subset) [10] (iii) they retain the ability to formtumors when they are injected into nonobese diabeticseverecombined immunodeficiency (NOD-SCID) mice and (iv)accumulating scientific evidence indicates that cancer stemcells express ABC transporters which confer chemoresistanceon this subset of cells [11]

Characteristic cell surface markers and the property ofCSCs to exclude certain fluorescent dyes can act as identifyingfeatures for stem cells Cancer stem cells are detected by anumber of cell surfacemarkers including CD133 (prominin 1)and CD166 both of which were already known to define stemand progenitor cells NESTIN (along with CD133) has alsobeen regarded as a CSC marker in disseminated tumor cellsof metastatic melanoma patients In C6 glioma cells it wasdemonstrated that a small fraction of cells that could formtumor spheres also expressed potential stem cell markersCD133 andNESTIN In conclusion it can be stated that CD133is an indicator but may not be a reliable marker for definingCSCs in solid tumors since it does not characterize tumor-initiating cells exclusively and that CD133 is a necessary albeitinsufficient criterion to identify CSCs in solid tumors [12]ABC transporters such as ABCB1 ABCG2 and ABCB5 can

BioMed Research International 3

also serve as markers of CSCs ABCB5 expression in tumorcells was seen to correlatewith clinicalmelanomaprogressionand a subpopulation of human melanoma cells was observedto coexpress ABCB1 ABCB5 and ABCC2 in addition tostem cell markers [12] Additionally it was found that tumor-initiating cells in humanmelanoma could be identified by theexpression of ABCG2 coexpressedwith CD133 [12] Howeverit must be kept in mind that tumor-initiating stem cells arefor the most part heterogeneous and a specific markerset ofmarkers has not been found to identify CSCs in solid tumors[12]

5 ABC Transporters Define the SidePopulation in Stem Cells

The side population (SP) of CSCs is a subset of stem cellsthat have a high capability for effluxing antimitotic drugsCells making up the side population can be isolated bytheir capacity to efflux fluorescent dye Hoechst 33342 orrhodamine 123 with the help of a flow cytometer Thisparticular population is called the ldquoside populationrdquo becauseduring flow-cytometry analysis these cells can be visualizedas a negatively stained population off to ldquothe siderdquo of themainpopulation of cells [13]The current understanding is that thedrug transporting capability of these cells is likely conferredby certain ABC transporters including ABCB1 (rhodamine123) and ABCG2 (Hoechst 33342) Side population (SP)isolation from a number of cancer cell lines has shownthat the expression of ABCB1 is upregulated in the SPcompared to the normal population SP cells isolated fromthe PANC1 pancreatic cancer cell line have been found toexpress both ABCB1 and ABCG2 [14] The isolated SP cellsconstituted around 21ndash87 of the total population of viablecells identified by Hoechst 33342 staining This populationwas found to have an enhanced capacity for the efflux ofHoechst dye that was postulated to be due to ABCB1 andABCG2 expression Targeting this side population thus mayprovide an alternate approach to cancer therapy

6 ABC Transporters as a Causal Determinantof Drug Resistance in Cancer Stem Cells

Clinical drug resistance as we now understand is mul-tifactorial involving alteration in drug targets inactiva-tiondetoxification of the drug decreased drug uptakeincreased drug efflux and the dysregulation of apoptoticpathways [1] ABC transporters including ABCG2 ABCB1and ABCC1 to name but a few are known to be associatedwith drug resistance [3] ABCG2 is a half transporter postu-lated to function as a homodimer It has amolecular weight of72 kD and a particularly broad substrate-specificity It has thecapacity to transport doxorubicin mitoxantrone topotecanmethotrexate and tyrosine kinase inhibitors among othersAnother important transporter is ABCB1 (also known as P-glycoprotein) Its expression has been found in over 50 ofall drug resistant tumors Human ABCB1 is the product ofthe MDR1 gene and acts as an ATP dependent pump for avariety of hydrophobic compounds including anticancer and

antimicrobial drugs Side population isolation from cancercell lines has demonstrated that the expression of ABCB1is upregulated in the SP when compared to the normalpopulation SP cells isolated from the PANC1 pancreaticcancer cell line have been found to express both ABCB1 andABCG2 [3] The isolated side population cells were around21ndash87 of the total population of viable cells identifiedby Hoechst 33342 staining This side population had anenhanced capacity for the efflux of Hoechst dye which waspostulated to be due to ABCB1 and ABCG2 expression [3]However apart fromABC transporters there are other factorsthat may determine drug resistance in cancer stem cells suchas the capacity of a stem cell for DNA repair and its quiescentstate Usually tumors which recur after an initial response tochemotherapeutic drugs become resistant to multiple drugsThe cancer stem cell model of drug resistance postulatesthat the original tumor would contain a small populationof tumor stem cells and their differentiated progeny Afterexposure to the drug only tumor stem cells (expressingdrug transporters) would survive These stem cells couldthen divide and repopulate the tumor with stem cells anddifferentiated cells [13]

7 Strategies for Targeting ABC Transportersin Cancer Stem Cells

Cancer stem cells as we know retain the ldquoexclusiverdquo propertyto drive the growth and spread of a tumor These cancerstem cells express a number of ABC transporters includingABCG2 ABCB1 ABCB5 andABCC1 An important strategyto overcome drug resistance in CSCs would be to targetthe functioning of ABC transporters in these cells We willdiscuss the current strategies for inhibiting the function ofthese transporters that can be applied to CSCsThe strategiesare as follows

(i) Chemotherapy through competitive and allostericmodulators

(ii) Chemotherapy mediated by nanoparticle targeting(iii) Targeting transcriptional regulation of ABC trans-

porters(iv) MicroRNA therapeutics(v) Targeting signaling pathways involved in the regula-

tion of ABC transporters(vi) Combinational targeting with CSC targeting agents

and transporter modulating drugs or dual targetingwith a single agent

(i) Chemotherapy through Competitive and Allosteric Modula-tors A directed effort has been devoted to the developmentof inhibitors against ABC efflux pumps These inhibitorscan be classified as competitive inhibitors that bind tothe substrate site and actively compete with the substrateand allosteric modulators that bind at a region that is notthe substrate-binding site but can cause a conformationalchange in the transporter that affects the functioning ofthe transporter Historically we can trace the journey of


competitive inhibitors from development to clinical appli-cation First-generation inhibitors include drugs like vera-pamil and cyclosporine that could inhibit the ABCB1 effluxpump However the low efficacy of these drugs in theclinical setting necessitated the development of rationallydesigned inhibitors based on a quantitative structural activityrelationship (QSAR) These were termed second-generationcompounds and included PSC833 and biricodar (VX-710)However it was discovered that PSC833 can significantlyreduce the clearance of chemotherapeutics and as a resultcould elevate toxicity of the drug [15] Thus the clinical trialswith these second-generation inhibitors were largely negativeprobably due to pharmacokinetic interactions between thechemotherapeutics and the P-gp inhibitor Development ofthird-generation inhibitors was thus necessitated that couldpossibly overcome the drawbacks of the previous genera-tion XR9576 (tariquidar) and LY335979 (zosuquidar) weretwo such drugs that were developed XR9576 inhibit bothABCB1 and ABCG2 while LY335979 is a specific inhibitorof ABCB1P-glycoprotein Another potential inhibitor isFumitremorgin C (FTC) that proved to be a very potentinhibitor of ABCG2 However a major drawback of thisparticular drug was undesirable neurotoxic effects Naturalproducts have also been shown to have inhibitory effects onABC transporters including ABCB1 ABCC1 and ABCG2These include the polyphenols and curcumin that appear tomodulate the effects of ABCB1 ABCC1 and ABCG2 [15]Another promising modulator is NSC 73306 a thiosemi-carbazone derivative It has been shown to be selectivelycytotoxic to cells that overexpress ABCB1P-glycoprotein[16] It was also discovered that NSC73306 could inhibitABCG2 mediated drug resistance to both mitoxantrone andtopotecan It was subsequently postulated that NSC73306may have a dual mode of action by eliminating both P-gpexpressing cells while also being a potent modulator thatcould resensitize ABCG2 overexpressing cells to chemother-apeutics [17] Traditionally competitive inhibitors are usedto overcome drug resistance however it was shown thatthe efflux of mitoxantrone from the leukemic stem cellscould not be efficiently inhibited by the ABCB1 inhibitorsverapamil and PSC-833 The researchers hypothesized thatdifferences between leukemic and normal stem cells mayhave been caused by additional transport mechanisms inthe leukemic stem cells [18] Another approach to the prob-lem of overcoming drug resistance in CSCs would be toutilize allosteric modulators that would not compete withthe substrate but would potentially cause a conformationalchange in the transporter that would affect its functionIt has recently been shown that allosteric modulators oftransporters may have the potential to inhibit their function-ing [19ndash23] A group of compounds known as flupentixolsthat include cis(Z) flupentixol and trans(E) flupentixol havebeen found to allosterically inhibit the transporter ABCB1[19ndash23] Studies have indicated that there is evidence oftwo modulator-specific sites at the lipid protein interfaceof ABCB1 (P-gp) that demonstrate negative synergy ininfluencingATP hydrolysis [19]These inhibitorsmodulatorshave the potential to target ABC transporters in cancer stemcells

(ii) Chemotherapy Mediated by Nanoparticle Targeting Con-ventional chemotherapy has several drawbacks among whichis the fact that the drug cannot be specifically targeted tothe tumor and also the problem of drug efflux by ABCtransporters These issues can partially be addressed with theuse of nanoparticles Nanoparticles (NPs) are widely used indrug delivery systems One or more cytotoxic drugs may beencapsulated within or bound to a nanosphere or nanocap-sule that generally has a diameter between 1 and 1000 nm [24]These nanospheres are usually composed of a semisyntheticbiodegradable polymer PLGA (polylactide-co-glycolide) isone polymer that has been approved by the FDA Other kindsof nanocarriers may be micelles liposomes carbon nan-otubules and so forth Nanodrug delivery systems (NDDS)reach the tumor cell through molecular-targeted delivery torelease the cargo to the affected cell or across the cell mem-brane and ideally should exhibit very low to zero off-targeteffects [25 26] There have been some studies that supportthe use of nanoparticles to combat drug resistance It wasobserved that there was a significant reduction in tumor sizeand increased animal survival rate in the rat xenograft gliomamodel with indomethacin loaded nanocapsules [27] It wasalso discovered that Paclitaxel loaded PLA-PEG (polyethy-lene glycol) ligand conjugated nanoparticles enhanced drugaccumulation in MCF-7 cell tumor xenograft model [28] Itwas also indicated that nanoparticles loaded with dual drugsseem to have a better therapeutic efficacy Studies have shownthe role of curcumin as a drug resistancemodulator enhancedthe therapeutic potential of nutlin-3a for targeting multidrugresistant tumors [29] suggesting that loading nanoparticleswith both curcumin and nutlin-3a may be more effective Ithas been proposed that ABCB1P-glycoprotein functions asa ldquohydrophobic vacuum cleanerrdquo pumping out drugs fromthe lipid bilayer itself before the drug can reach the cyto-plasm [30] In this context ABCB1P-glycoprotein mediatedefflux may be circumvented by coadministration of P-gpinhibitors and anticancer drugs packaged in nanoparticleswhich have the property to evade P-gp recognition at cellmembrane As a result these nanoparticles have the capabilityto deliver a drug directly into the cytoplasm Several studieshave shown that this strategy may effectively circumventtransporter-mediated drug efflux Paclitaxel a P-gp substrateand verapamil a P-gp inhibitor were encapsulated in PLGAnanoparticles and this could circumvent P-gp-mediated drugefflux in MDR tumor cells [31] The same group also demon-strated that doxorubicin loaded nanoparticles were able toincrease the cellular delivery and therapeutic efficacy of P-gpsubstrates in P-gp overexpressing cells [32]

It was very recently shown that a doxorubicin-encapsu-lated polymeric nanoparticle surface (decorated with chi-tosan) could specifically target the CD44+ receptors of stem-like cells in 3D mammary spheroids in xenograft tumormodels This particular design of nanoparticles was seento increase the cytotoxicity of the doxorubicin 6-fold incomparison to the use of free doxorubicin [33] Judgingfrom the initial success of the nanoparticle mediated deliverysystem it would be pertinent to assume that this system mayin the future be applied to other tumors exhibiting drugresistance in stem cells


(iii) Targeting Transcriptional Regulation of ABCTransportersAnother approach to combating drug resistance in CSCswould be to target the transcriptional regulation of ABCtransporters Efforts to combat drug resistance caused byABC efflux transporters have mainly focused on the use offunctional modulators rather than on therapeutic targetingof transcription Developing drug candidates that couldpotentially inhibit ABC transporters at the transcriptionallevel would be an effective mechanism of avoiding drugresistance and could also be an excellent approach to targetCSCs which overexpress these efflux pumps It may bepossible to prevent transcriptional activation with the aidof prophylactic intervention Ecteinascidin 743 (ET-743) cantarget the transcriptional activation of P-gp (ABCB1MDR1)in the laboratory This compound is a natural product(tris)tetrahydroisoquinoline related to the saframycin familyof compounds isolated from the sea squirt Ecteinascidiaturbinata ET743 is capable of interfering with the activationof MDR1 transcription and basic studies have shown thatit may affect the MDR1 enhanceosome complex howeverits precise mechanism of action has not been elucidated[34] However advances in the elucidation of transcriptionalregulation of ABC transporters may pave the way toward thedevelopment of novel therapeutic agents Overexpression ofMDR1 in drug resistant cells may be a result of gene ampli-fication or transcriptional overexpression It was discoveredthat p53 may have a role in the regulation of the MDR1 geneWild-type p53 was found to repress transcription of bothendogenous MDR1 gene and MDR1 reporter constructs [35]through direct DNA binding at the HT site Different p53family members such as p63 and p73 were found to activateMDR1 transcription through an indirect interaction with theAPE site (the alternative p63p73 element) indicating that p53DNA binding domains can differentially regulate transcrip-tion through both DNA binding dependent and independentmechanisms [36] C-terminal-binding protein 1 (CtBP-1) canalso act as an activator ofMDR1 gene transcription and couldbe a new target for inhibitingMDR1-mediated drug resistance[37] MDR1 gene expression may be activated by differentmeans such asUV irradiation sodium butyrate retinoic acidHDAC inhibitors and certain chemotherapeutics Signalsfrom different stimuli may converge on a region of theMDR1 promoter which has been referred to as the ldquoMDR1enhanceosomerdquo [34] thus the enhanceosome would makean attractive therapeutic target The enhanceosome containsbinding sites for a variety of transcription factors Theseproteins can recruit PCAF (a histone acetyl transferase) tothe MDR1 promoter regionThe result of PCAF recruitmentwould be acetylation of promoter-proximal histones followedby transcriptional activity If the complex mechanistic pro-cess of transcription regulation can be understood there ispotential to develop agents that prevent the transcriptionalactivity of a variety of drug transporters There were a largenumber (232) of potential regulatory transcription factorbinding sites discovered through in silico studies in thepromoter region of ABCB5 including CREB PAX6 CEBPand OCT1 and also ABCC1 [3] Functional validation ofthese transcription factors in ABCB5 and other transporters

would be an interesting approach to initiate studies to developpotential inhibitory agents

(iv) MicroRNA Therapeutics MicroRNAs (miRNAs) areclassified as small single stranded RNAs that range from19 to 25 nucleotides in length and are noncoding Theyhave the capacity to regulate gene expression by usuallybinding to the 31015840UTR An ldquoindirectrdquo or ldquopotentialrdquo modelhas been proposed that links miRNAs to the regulation ofCSCs [38] This model proposed states that the aberrantexpression of microRNA (oncomiRs oncogenic miRNA ortumor suppressor miRNA) may result in the dysregulationof certain stem cell genes [38] The impact of this aberrantexpression might be to cause increased self-renewal of CSCsand impaired differentiation of a subset of CSCsThe authorspropose that this dysregulation could result in carcinogenesisand oncogenesis [38] A few miRNAs have been found thatregulate ABCB1 and ABCG2 and could be important toABCB1 and ABCG2 expression in CSCs hsa-miR-451 wasfound to regulate ABCB1 in MCF7 breast cancer cells [39]It was also discovered that both miR-451 and miR-27a couldregulate ABCB1 expression in multidrug resistant A2780DX5and KB-V1 cancer cell lines [40] It was also observed thaton treating K562 human chronic myelogenous leukemia cellswith increasing concentrations of imatinib an inverse regula-tion of ABCG2 expression was seen with both miR-328 andmiR-212 [41] In a reporter gene assay miR-212 was definitelyshown to inversely regulate ABCG2 [41] given that ABCG2 isexpressed in CSCrsquos this could be a very important finding forfuture therapies From microRNA profiling in drug sensitiveand drug resistantMCF-7 breast cancer cells it was discoveredthat there was a differential expression of miRNA betweenthese cells Notably hsa-miR-382 hsa-miR-23b and hsa-miR-885-5p were upregulated and hsa-mir-218 hsa-miR-758 andhsa-miR-548d-5p were downregulated [42] An additionalstudy documented that in human breast cancer cells resistantto etoposide (MCF-7VP-16) miR-326 was downregulatedin resistant cells Subsequently transfection of miR-326 intoMCF-7VP-16 cells downregulated ABCC1 expression andincreased the sensitivity to etoposide and doxorubicin [43]suggesting that there are possible microRNA targets thatcan be investigated to circumvent ABC transporter-mediateddrug resistance in CSCs

(v) Targeting Signaling Pathways Involved in the Regulation ofABC Transporters Regulating the protein expression of ABCtransporters in CSCs may provide an alternative strategy totarget ABC transporter functioning in CSCs The Hedgehog(Hh) pathway is involved in several developmental processesof cells such as the determination of cell fate patterningproliferation survival anddifferentiation Inmammals threeHh proteins (Sonic Indian and Desert) are present Hhacts by binding to Patched (PTCH) [44] In the absenceof ligand PTCH constitutively represses the activity of aprotein called Smoothened (SMO)WhenHh ligand binds toPTCH the repression of SMO is released and the expressionandor posttranslational processing of the three GLI zinc-finger transcription factors is achieved The Gli proteins arecapable of inducing the expression of several target genes


[45] It has been indicated that Hedgehog signaling canregulate the expression ofMDR1 andABCG2 [46] Treatmentof PC3 cells with cyclopamine a SMO signaling elementinhibitor downregulates the expression levels of MDR1 andABCG2 Targeting of cancer stem cells holds great promisein treating aggressive cancers Diverse pathways are involvedin stem cell differentiation and renewal including oncogeniccascades like EGFR hedgehog (HH) WNT-120573-catenin anda variety of oncogenic signaling elements including NF-KB AKT PI3 kinase Cox 2 and ABC efflux pumps [4748] These factors play a key role in regulating SC self-renewal survival differentiation and drug resistance andmay be viable candidates for molecular targeting It wasrecently found that abnormal expression of the hedgehog(Hh) signaling pathway transcription factor Gli1 is involvedin the regulation of ABC transporters ABCB1 and ABCG2 inovarian cancer [49] It was found that the inhibition of Gli1expression can decrease ABCB1 and ABCG2 gene expressionlevels and enhance the response of ovarian cancer cells tospecific chemotherapeutics [49] Thus targeting signalingpathways may provide a directed approach to overcomingdrug resistance in CSCs

(vi) Combinational Targeting with CSC Targeting Agents andTransporter Modulating Drugs or Dual Targeting with a SingleAgent Another possible avenue to target CSCs may be witha single compound that targets CSCs and also modulatesABC transporters or with a combination of two differenttypes of drug In the case of two drugs this could lead toa potentiated effect of the combination of agents A largefamily of polyphenolic molecules called flavenoids modulatemultidrug resistant transporters and also inhibit CSC growthThe anticancer properties of flavenoids are primarily due totheir antimitotic activity and also due to inhibition of certainenzymes Flavonoids may also inhibit the function of ABCtransporters such as MDR1P-gp MRPs and ABCG2BCRPFlavenoids are ideal for interaction with these pumps becausethey are hydrophobic molecules and they display low toxicitybut show a broad spectrum of biological activities One suchcompound is LY294002 It is a PI3K specific inhibitor andis able to block the osteosarcoma CSC cell cycle (G0G1)through inducing apoptosis by preventing phosphorylationof PKBAkt via PI3K phosphorylation inhibitionThis partic-ular compound also inhibits BCRP ABCB1P-gp and MRP1[44]

8 Conclusions

It is imperative to gain a better understanding of themechanisms involved in the resistance of stem cells tochemotherapy Once the mechanism has been understoodit can lead to the discovery of new therapeutic targets andimprovement of current anticancer strategies One factorthat is responsible for chemoresistance in CSCs is ABCtransporters Based on numerous studies it is apparent thattargeting ABC transporters in CSCs can lead to a betteroutcome for patients given that according to the cancer stemcell hypothesis these stem cells are the only cells in thetumor capable of giving rise to a new tumor The stem cell

model of drug resistance is an important step forward in thefield of cancer drug resistance because it gives us an avenueto explain how cancers which show an apparent completeclinical response to chemotherapy can relapse either withinmonths or even years later In order to elucidate and improveupon this model however it is necessary to define stem cellsby their long-term self-renewal potential and not just by theexistence of a side population The simple fact that we cannow identify purify and propagate cancer stem cells mayin the future allow us to develop new strategies to improvetargeted cancer therapeutics and thereby improve patientoutcomes

Conflict of Interests

The author declares that there is no conflict of interestsregarding the publication of this paper

References

[1] M M Gottesman ldquoMechanisms of cancer drug resistancerdquoAnnual Review of Medicine vol 53 pp 615ndash627 2002

[2] M D Hall M D Handley andMM Gottesman ldquoIs resistanceuseless Multidrug resistance and collateral sensitivityrdquo Trendsin Pharmacological Sciences vol 30 no 10 pp 546ndash556 2009

[3] K Moitra H Lou and M Dean ldquoMultidrug efflux pumps andcancer stem cells insights into multidrug resistance and ther-apeutic developmentrdquo Clinical Pharmacology andTherapeuticsvol 89 no 4 pp 491ndash502 2011

[4] M Shackleton ldquoNormal stem cells and cancer stem cells similarand differentrdquo Seminars in Cancer Biology vol 20 no 2 pp 85ndash92 2010

[5] N A Lobo Y Shimono D Qian andM F Clarke ldquoThe biologyof cancer stem cellsrdquo Annual Review of Cell and DevelopmentalBiology vol 23 pp 675ndash699 2007

[6] M DeanThe Human ATP-Binding Cassette (ABC) TransporterSuperfamily National Center for Biotechnology InformationBethesdaMdUSA 2002 httpwwwncbinlmnihgovbooksNBK3

[7] C W Scharenberg M A Harkey and B Torok-Storb ldquoTheABCG2 transporter is an efficient Hoechst 33342 efflux pumpand is preferentially expressed by immature human hematopoi-etic progenitorsrdquo Blood vol 99 no 2 pp 507ndash512 2002

[8] A H Schinkel J J M Smit O van Tellingen et al ldquoDisruptionof the mouse mdr1a P-glycoprotein gene leads to a deficiency inthe blood-brain barrier and to increased sensitivity to drugsrdquoCell vol 77 no 4 pp 491ndash502 1994

[9] L Tang SM Bergevoet C Gilissen et al ldquoHematopoietic stemcells exhibit a specific ABC transporter gene expression profileclearly distinct from other stem cellsrdquo BMC Pharmacology vol10 article 12 2010

[10] S M Graham H G Joslashrgensen E Allan et al ldquoPrimitivequiescent Philadelphia-positive stem cells from patients withchronic myeloid leukemia are insensitive to STI571 in vitrordquoBlood vol 99 no 1 pp 319ndash325 2002

[11] C Hirschmann-Jax A E Foster G G Wulf et al ldquoA distinctlsquoside populationrsquo of cellswith high drug efflux capacity in humantumor cellsrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 101 no 39 pp 14228ndash142332004


[12] Y Welte J Adjaye H R Lehrach and C R A RegenbrechtldquoCancer stem cells in solid tumors elusive or illusiverdquo CellCommunication and Signaling vol 8 article 6 2010

[13] M Dean T Fojo and S Bates ldquoTumour stem cells and drugresistancerdquo Nature Reviews Cancer vol 5 no 4 pp 275ndash2842005

[14] J Zhou C-YWang T Liu et al ldquoPersistence of side populationcells with high drug efflux capacity in pancreatic cancerrdquoWorldJournal of Gastroenterology vol 14 no 6 pp 925ndash930 2008

[15] C-P Wu A M Calcagno and S V Ambudkar ldquoReversalof ABC drug transporter-mediated multidrug resistance incancer cells evaluation of current strategiesrdquoCurrentMolecularPharmacology vol 1 no 2 pp 93ndash105 2008

[16] J A Ludwig G Szakacs S E Martin et al ldquoSelective toxicityof NSC73306 in MDR1-positive cells as a new strategy tocircumvent multidrug resistance in cancerrdquo Cancer Researchvol 66 no 9 pp 4808ndash4815 2006

[17] C-P Wu S Shukla A M Calcagno M D Hall M MGottesman and S V Ambudkar ldquoEvidence for dual mode ofaction of a thiosemicarbazone NSC73306 a potent substrate ofthe multidrug resistance linked ABCG2 transporterrdquoMolecularCancer Therapeutics vol 6 no 12 pp 3287ndash3296 2007

[18] M H G P Raaijmakers E P L M de Grouw B A vander Reijden T J M de Witte J H Jansen and R A PRaymakers ldquoABCB1 modulation does not circumvent drugextrusion from primitive leukemic progenitor cells and maypreferentially target residual normal cells in acute myelogenousleukemiardquo Clinical Cancer Research vol 12 no 11 I pp 3452ndash3458 2006

[19] D Mandal K Moitra D Ghosh D Xia and S Dey ldquoEvidencefor modulatory sites at the lipid-protein interface of the humanmultidrug transporter P-glycoproteinrdquo Biochemistry vol 51 no13 pp 2852ndash2866 2012

[20] S Dey M Ramachandra I Pastan M M Gottesman and SV Ambudkar ldquoEvidence for two nonidentical drug-interactionsites in the human P-glycoproteinrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 94 no20 pp 10594ndash10599 1997

[21] C PascaudMGarrigos and SOrlowski ldquoMultidrug resistancetransporter P-glycoprotein has distinct but interacting bindingsites for cytotoxic drugs and reversing agentsrdquo BiochemicalJournal vol 333 no 2 pp 351ndash358 1998

[22] CMartin G Berridge C F Higgins P Mistry P Charlton andR Callaghan ldquoCommunication between multiple drug bindingsites on P-glycoproteinrdquoMolecular Pharmacology vol 58 no 3pp 624ndash632 2000

[23] N Maki P Hafkemeyer and S Dey ldquoAllosteric modulation ofhuman P-glycoprotein Inhibition of transport by preventingsubstrate translocation and dissociationrdquoThe Journal of Biolog-ical Chemistry vol 278 no 20 pp 18132ndash18139 2003

[24] Q Yin J Shen Z Zhang H Yu and Y Li ldquoReversal of mul-tidrug resistance by stimuli-responsive drug delivery systemsfor therapy of tumorrdquo Advanced Drug Delivery Reviews vol 65no 13-14 pp 1699ndash1715 2013

[25] S Moghassemi and A Hadjizadeh ldquoNano-niosomes as nano-scale drug delivery systems an illustrated reviewrdquo Journal ofControlled Release vol 185 no 1 pp 22ndash36 2014

[26] LGuoH Zhang FWang et al ldquoTargetedmultidrug-resistancereversal in tumor based onPEG-PLL-PLGApolymer nano drugdelivery systemrdquo International Journal of Nanomedicine vol 10pp 4535ndash4547 2015

[27] A Bernardi E Braganhol E Jager et al ldquoIndomethacin-loadednanocapsules treatment reduces in vivo glioblastoma growth ina rat glioma modelrdquo Cancer Letters vol 281 no 1 pp 53ndash632009

[28] Y Patil T Sadhukha L Ma and J Panyam ldquoNanoparticle-mediated simultaneous and targeted delivery of paclitaxeland tariquidar overcomes tumor drug resistancerdquo Journal ofControlled Release vol 136 no 1 pp 21ndash29 2009

[29] M Das and S K Sahoo ldquoFolate decorated dual drug loadednanoparticle role of curcumin in enhancing therapeutic poten-tial of nutlin-3a by reversing multidrug resistancerdquo PLoS ONEvol 7 no 3 Article ID e32920 2012

[30] C F Higgins and M M Gottesman ldquoIs the multidrug trans-porter a flippaserdquo Trends in Biochemical Sciences vol 17 no 1pp 18ndash21 1992

[31] M D Chavanpatil Y Patil and J Panyam ldquoSusceptibil-ity of nanoparticle-encapsulated paclitaxel to P-glycoprotein-mediated drug effluxrdquo International Journal of Pharmaceuticsvol 320 no 1-2 pp 150ndash156 2006

[32] MD Chavanpatil A Khdair B Gerard et al ldquoSurfactant-poly-mer nanoparticles overcome P-glycoprotein-mediated drugeffluxrdquoMolecular Pharmaceutics vol 4 no 5 pp 730ndash738 2007

[33] W Rao H Wang J Han et al ldquoChitosan-decorateddoxorubicin-encapsulated nanoparticle targets and eliminatestumor reinitiating cancer stem-like cellsrdquo ACS Nano vol 9 no6 pp 5725ndash5740 2015

[34] KW Scotto and R A Johnson ldquoTranscription of themultidrugresistance geneMDR1 a therapeutic targetrdquoMolecular Interven-tions vol 1 no 2 pp 117ndash125 2001

[35] K-V Chin K Ueda I Pastan and M M Gottesman ldquoModu-lation of activity of the promoter of the human MDR1 gene byRas and p53rdquo Science vol 255 no 5043 pp 459ndash462 1992

[36] R A Johnson E M Shepard and K W Scotto ldquoDifferentialregulation of MDR1 transcription by the p53 family membersrole of the DNA binding domainrdquo The Journal of BiologicalChemistry vol 280 no 14 pp 13213ndash13219 2005

[37] W Jin K W Scotto W N Hait and J-M Yang ldquoInvolvementof CtBP1 in the transcriptional activation of the MDR1 gene inhuman multidrug resistant cancer cellsrdquo Biochemical Pharma-cology vol 74 no 6 pp 851ndash859 2007

[38] J T Desano and L Xu ldquoMicroRNA regulation of cancer stemcells and therapeutic implicationsrdquo The AAPS Journal vol 11no 4 pp 682ndash692 2009

[39] O Kovalchuk J Filkowski J Meservy et al ldquoInvolvementof microRNA-451 in resistance of the MCF-7 breast cancercells to chemotherapeutic drug doxorubicinrdquoMolecular CancerTherapeutics vol 7 no 7 pp 2152ndash2159 2008

[40] H Zhu H Wu X Liu et al ldquoRole of MicroRNA miR-27a andmiR-451 in the regulation of MDR1P-glycoprotein expressionin human cancer cellsrdquo Biochemical Pharmacology vol 76 no5 pp 582ndash588 2008

[41] E Turrini S Haenisch S Laechelt T Diewock O Bruhnand I Cascorbi ldquoMicroRNA profiling in K-562 cells underimatinib treatment influence of miR-212 and miR-328 onABCG2 expressionrdquo Pharmacogenetics and Genomics vol 22pp 198ndash205 2012

[42] K Moitra K Im K Limpert et al ldquoDifferential gene andmicroRNA expression between etoposide resistant and etopo-side sensitive MCF7 breast cancer cell linesrdquo PLoS ONE vol 7no 9 Article ID e45268 2012


[43] Z Liang H Wu J Xia et al ldquoInvolvement of miR-326 inchemotherapy resistance of breast cancer through modulatingexpression of multidrug resistance-associated protein 1rdquo Bio-chemical Pharmacology vol 79 no 6 pp 817ndash824 2010

[44] L Zinzi M Contino M Cantore E Capparelli M Leopoldoand N A Colabufo ldquoABC transporters in CSCs membranesas a novel target for treating tumor relapserdquo Frontiers inPharmacology vol 5 article 163 2014

[45] H Sasaki Y Nishizaki C-C Hui M Nakafuku and HKondoh ldquoRegulation of Gli2 and Gli3 activities by an amino-terminal repression domain implication of Gli2 and Gli3 asprimary mediators of Shh signalingrdquoDevelopment vol 126 no17 pp 3915ndash3924 1999

[46] J Sims-Mourtada J G Izzo J Ajani and K S C Chao ldquoSonicHedgehog promotes multiple drug resistance by regulation ofdrug transportrdquo Oncogene vol 26 no 38 pp 5674ndash5679 2007

[47] J R Remsberg H Lou S G Tarasov M Dean and N ITarasova ldquoStructural analogues of smoothened intracellularloops as potent inhibitors of Hedgehog pathway and cancercell growthrdquo Journal of Medicinal Chemistry vol 50 no 18 pp4534ndash4538 2007

[48] M Mimeault and S K Batra ldquoRecent progress on tissue-resident adult stem cell biology and their therapeutic implica-tionsrdquo Stem Cell Reviews vol 4 no 1 pp 27ndash49 2008

[49] Y Chen MM Bieber and N N H Teng ldquoHedgehog signalingregulates drug sensitivity by targeting ABC transporters ABCB1andABCG2 in epithelial ovarian cancerrdquoMolecular Carcinogen-esis vol 53 no 8 pp 625ndash634 2014

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Drug DeliveryJournal of



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Emergency Medicine InternationalHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Autoimmune Diseases


Anesthesiology Research and Practice

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MEDIATORSINFLAMMATION

of


2 The Cancer Stem Cell Model

The cancer stem cell (CSC) model expounds on the processby which established cancers are able to propagate [4] TheCSC theory proposes that a defined population of cancer cells(known as cancer stem cells) have the ldquoexclusiverdquo propertyto drive the growth and propagation of a tumor CSCs canalso give rise to progeny that have a limited ability to divide[5] However tumorigenic stem cells frequently lack some ofthe control mechanisms present in normal stem cells whereproliferation is tightly regulated and the genomic integrity ofthe cells is maintained If the doctrine that tumors containstem cells is assumed to be correct then we could possiblyinterpret the accumulation of mutations in these stem cells asthe basic ldquomultisteprdquo process of carcinogenesis The uncannyability of cancer stem cells to resist chemo- and radiotherapywould lead us to ask the question Does the ldquoinnaterdquo orldquointrinsicrdquo resistance of stem cells to radiation and toxinscontribute to the failure of some cancer therapies Andultimately askHow canwe exploit our knowledge of stem cellbiology to specifically target CSCrsquos in order to overcome drugresistance and improve therapeutic outcome in the clinic

3 ABC Transporters and Stem Cells

ATP-binding cassette transporters (ABC transporters) areintricate molecular pumps most of which catalyze the trans-port of awide array of substrates across biologicalmembranesagainst a concentration gradient by the hydrolysis of ATPThe human genome is known to encode 48 ABC transportergenes categorized into seven subfamilies ranging from Ato G [6] ABC transporters are classified based on thesequence and organization of their ATP-binding domain(s)that contain specific conserved motifs Walker A andWalkerB (present in all ATP-binding protein) however ABC trans-porters contain an additionalmotif the signaturemotif or theC-loop that is located upstreamofWalker Bmotif Functionaltransporters usually contain two transmembrane domains(TMDs) generally made up of 6ndash12 membrane-spanningalpha-helices that are primarily responsible for determiningsubstrate specificity Additionally there are two nucleotidebinding domains (NBDs) that bind and hydrolyze ATPproviding the energy for substrate translocation (Figure 1)ABC transporters may be expressed in stemprogenitor cellsderived from several types of normal tissue and also inhematopoietic cells Hematopoietic stem cells (HSCs) werefound to express high levels of ABCG2 andor ABCB1transporters [7] Mouse knockouts for ABCB1 ABCG2 orABCC1 revealed that mice are particularly sensitive to somecompounds such as mitoxantrone vinblastine ivermectinand topotecan indicating that these transporters may havea role in protecting the stem cells from toxic substances [8]Using the aid of Taqman low density arrays ABC transporterswere also found to be expressed in normal stem cells such ashematopoietic stem cells (HSCs) unrestricted somatic stemcells (USSCs) mesenchymal stem cells (MSCs) andmultipo-tent adult progenitor cells (MAPCs) [9] HSCs seemed to relyon a different repertoire of these transporters compared toother tissuecell types based on the fact that gene signatures

TMD 2

NBD 1 NBD 2

TMD 1

N

C

Figure 1 Schematic diagram of a typical ABC transporter(ABCB1P-glycoprotein) depicting the structural organization of afull transporter A full transporter typically contains 2 transmem-brane domains (TMDs) and 2 nucleotide binding domains (NBDs)

for ABC transporters were found to be radically differentbetween HSCs and other types of stem cells 16 transportersin total including ABCB1 and ABCG1 were discovered tobe consistently expressed at higher levels in HSCs whencompared to other transporters The transporters ABCA4ABCA8 ABCC9 and ABCG4 were consistently detected inMSCs and USSCs [9]

4 Distinguishing Characteristics andBiomarkers of CSCs

Cancer stem cells (CSCs) possess the unique property toevade radiotherapy and chemotherapy Compared to differ-entiated tumor cells CSCs have 4 distinguishing featuresthey are as follows (i) they are relatively quiescent (ii) theyhave a slow cycling rate (the relative quiescence and slowcycling rate afford these cells protection against chemother-apeutics that target rapidly dividing cells rather than a slowcycling subset) [10] (iii) they retain the ability to formtumors when they are injected into nonobese diabeticseverecombined immunodeficiency (NOD-SCID) mice and (iv)accumulating scientific evidence indicates that cancer stemcells express ABC transporters which confer chemoresistanceon this subset of cells [11]

Characteristic cell surface markers and the property ofCSCs to exclude certain fluorescent dyes can act as identifyingfeatures for stem cells Cancer stem cells are detected by anumber of cell surfacemarkers including CD133 (prominin 1)and CD166 both of which were already known to define stemand progenitor cells NESTIN (along with CD133) has alsobeen regarded as a CSC marker in disseminated tumor cellsof metastatic melanoma patients In C6 glioma cells it wasdemonstrated that a small fraction of cells that could formtumor spheres also expressed potential stem cell markersCD133 andNESTIN In conclusion it can be stated that CD133is an indicator but may not be a reliable marker for definingCSCs in solid tumors since it does not characterize tumor-initiating cells exclusively and that CD133 is a necessary albeitinsufficient criterion to identify CSCs in solid tumors [12]ABC transporters such as ABCB1 ABCG2 and ABCB5 can




























8 Conclusions





References
























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of




























8 Conclusions





References
























































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of













































Volume 2014

ToxinsJournal of

VaccinesJournal of















AddictionJournal of






Autoimmune Diseases




Journal of


Pharmaceutics



of

Overcoming Multidrug Resistance in Cancer Stem Cells

Documents

Transcript of Overcoming Multidrug Resistance in Cancer Stem Cells