Lung-cancer-stem-cells--The-root-of-resistance_2016_Cancer-Letters

Mini-review

Lung cancer stem cells:The root of resistance

Lauren MacDonagh a ,Steven G.Gray a ,Eamon Breen b ,Sinead Cuffe a ,Stephen P.Finn a ,c ,Kenneth J.O’Byrne d ,Martin P.Barr a ,*

Thoracic Oncology Research Group,School of Clinical Medicine,Institute of Molecular Medicine,Trinity Centre for Health Sciences,St.James’s Hospital and Trinity College Dublin,Ireland

b Flow Cytometry Core Facility,Institute of Molecular Medicine,Trinity Centre for Health Sciences,St.James’s Hospital and Trinity College Dublin,Ireland

c Department of Histopathology,St.James’s Hospital an

d Trinity Colleg

e Dublin,Ireland

d Cancer &Ageing Research Program,Queensland University of Technology,Brisbane,Australia

A R T I C L E I N F O Article history:

Received 10December 2015

Received in revised form 6January 2016Accepted 7January 2016Keywords:Lung cancer

Cancer stem cells Chemotherapy Resistance Markers

A B S T R A C T

In the absence of speci?c treatable mutations,platinum-based chemotherapy remains the gold stan-dard of treatment for lung cancer patients.However,5-year survival rates remain poor due to the development of resistance and eventual relapse.Resistance to conventional cytotoxic therapies pres-ents a signi?cant clinical challenge in the treatment of this disease.The cancer stem cell (CSC)hypothesis suggests that tumors are arranged in a hierarchical structure,with the presence of a small subset of stem-like cells that are responsible for tumor initiation and growth.This CSC population has a number of key properties such as the ability to asymmetrically divide,differentiate and self-renew,in addition to having increased intrinsic resistance to therapy.While cytotoxic chemotherapy kills the bulk of tumor cells,CSCs are spared and have the ability to recapitulate the heterogenic tumor mass.The identi?cation of lung CSCs and their role in tumor biology and treatment resistance may lead to innovative targeted thera-pies that may ultimately improve clinical outcomes in lung cancer patients.This review will focus on lung CSC markers,their role in resistance and their relevance as targets for future therapies.

Introduction

Lung cancer and resistance

Lung cancer is the leading cause of cancer-related death world-wide and is classi?ed into two main subtypes;non-small cell lung cancer (NSCLC)which accounts for approximately 85%of all lung cancers and small-cell lung cancer (SCLC)which is diagnosed in 15%of cases [1,2].Histologically,NSCLC is further divided into three sub-types;adenocarcinoma,squamous-cell carcinoma and large cell carcinoma [3].Smoking is a major risk factor for lung cancer and is associated with all histological subtypes,in particular,squa-mous cell carcinomas.Adenocarcinoma on the other hand,is the predominant lung cancer subtype commonly seen in never smokers.Despite improved advances in the treatment of lung cancer,the 5-year survival rate remains low,largely due to the emergence of resistance prior to and during the course of treatment with che-motherapy and radiation therapy.This resistance to therapy represents a signi?cant clinical challenge in the treatment of lung cancer and contributes largely to disease progression,recurrence

and mortality [4,5].Despite intense efforts to overcome such re-sistance in lung cancer and other cancer types using novel agents,alone and in combination with chemo-and radiotherapy,the un-derlying mechanisms conferring this resistant phenotype in lung cancer remain largely unknown [6].It is now well established that CSCs constitute a unique subset of cells which are distinct from the bulk of tumor cells by their exclusive ability to perpetuate the growth of a malignant population of cells,inde?nitely.This may explain the ineffectiveness of many conventional therapies and patient relapse [7].These important clinical observations have stimulated intense interest in experimental approaches for further investigation of CSCs and their role in the treatment of drug resistant lung cancer.The cancer stem cell hypothesis

The CSC hypothesis is now a well-accepted and widely studied ?eld within oncology.Cellular heterogeneity is a histological hall-mark of many solid tumors and the CSC hypothesis suggests that this heterogeneity within a tumor is due to a hierarchical cell dynamic and the presence of a small subpopulation of cells that display the properties of normal somatic stem cells [8].These CSCs are multipotent,and have two key characteristics of stem cells;the abilities to self-renew and to differentiate,properties necessary for tumor initiation and progression (Fig.1A ).CSCs can

*Corresponding author.Tel.:+35318963620;fax:+35318963503.E-mail address:mbarr@stjames.ie ;barrma@tcd.ie (M.P.Barr).https://www.360docs.net/doc/0d3388140.html,/10.1016/j.canlet.2016.01.012

Cancer Letters 372(2016)147–156

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asymmetrically divide (Fig.1C ),which enables them to simultane-ously self-perpetuate and to generate differentiated progeny and give rise to heterogenic tumor,with a consistently maintained CSC sub-population [9,10].Although asymmetric division allows for both self-renewal and differentiated progeny,it does not allow the CSC subpopulation to expand.However,the stem cell niche has been shown to expand during development.Similarly,the CSC popula-tion expands during periods of stress,suggesting that they can also symmetrically divide (Fig.1B )[11–13].CSCs can rely on either asym-metric or symmetric division or a combination of the two (Fig.1D ).Previous studies have shown that a pure lung CSC population is capable of giving rise to a heterogenic progeny of CSCs and non-CSC cells through asymmetric cell division,whereas non-CSC lung cancer cells undergo only symmetric division resulting in a pure non-CSC cell population [14].

Cellular stress,such as chemotherapy treatment,can induce sym-metric division of rare CSC populations and apoptosis of non-CSC cells,therefore enriching the population [15].Symmetric division of the CSC population during initial cycles of chemotherapy may trigger relapse in the form of a chemoresistant tumor [16].Despite an initial response to anti-cancer treatments,chemotherapeutic treat-ment can lead to the symmetric propagation of a small subpopulation of drug-tolerant cells with stem cell features [17–20].Cisplatin treat-ment causes the lung CSC population to expand in patient ex-plants and in H460-xenografted nude mice.Similar studies have also shown side-population CSC expansion following treatment with cy-totoxic drugs such as 5’?uorouracil,an anti-metabolite commonly used in the treatment of several tumor types [21,22].

Lung cancer stem cells

Conventional anti-cancer therapies kill the bulk of the tumor,however,CSCs exhibit robust intrinsic resistance and survive therapy due to increased telomere length,activation of anti-apoptotic path-ways,increased membrane transporter activity and their ability to migrate and metastasize [23].

Telomeres are comprised of a repetitive G-rich sequence and an abundance of associated proteins that form an effective cap that pro-tects chromosome ends.Telomerase,also known as telomere terminal transferase,is a ribonucleoprotein complex that adds the telomere DNA sequence repeats to lengthen the telomere.It confers limitless proliferative capacity to cells through its ability to elon-gate telomeres [24,25].Telomerase activity is readily detected in most cancers but not in somatic tissues and is essential for stem cell in-tegrity and longevity [26].Increased telomere length has previously been shown to confer resistance to a number of anti-cancer thera-pies;inversely,shortened telomere length is associated with drug sensitivity [27].Telomerase inhibition enhanced the pro-apoptotic response to anti-cancer therapies resulting in delayed relapse fol-lowing chemotherapeutic treatment [27–29].Lung cancer stem cells display longer telomeres than their non-CSC counterparts.Treat-ment with the speci?c telomerase inhibitor,MST312,has a strong and preferential anti-proliferative effect on the lung CSC popula-tion in vitro and in vivo [14].Inhibition of telomerase using Imetelstat,either alone or in combination with Trastuzumab,decreases breast CSCs and inhibits their capacity to self-renew in HER2positive breast cancer cells [30].

Several mechanisms have been postulated to play a role in re-sistance,including impaired apoptotic machinery,increased DNA-repair mechanisms,up-regulation of multidrug resistance proteins and membrane e?ux transporters [31].Such mechanisms may be responsible,at least in part,for driving CSCs into a phenotype of reduced apoptotic cell death,which in turn forms the basis of tumor progression.ATP-binding cassette (ABC)transporters,such as p-glycoprotein and multidrug resistant associated protein (MRP1),are membrane transporters that can pump structurally unrelated small molecules,such as cytotoxic chemotherapeutic drugs out of the cell.Normal stem cells and lung CSCs express high levels of ABC-transporters resulting in low intracellular drug concentrations [32,33].For example,the ABCG2gene is highly expressed in hematopoi-etic and lung cancer stem cells but is switched off in most terminally differentiated progeny [34].

Cancer stem cells are chemoresistant and metastatic,two fea-tures that correlate with poor prognosis and tumor recurrence [35].Lung adenocarcinoma CSCs have increased expression of galectin-1,a multifunctional protein which promotes invasiveness and metastasis and is associated with poor overall survival and lymph node metastasis [36].Epithelial to mesenchymal transition (EMT)is an evolutionary conserved developmental process.Studies indi-cate that metastatic cancer cells,cells which have undergone EMT,exhibit a CSC phenotype [37,38].Numerous pathways,including the Wnt/β-catenin [39–42],Notch [43,44],Hedgehog [45–47]and NFκB [48]regulate CSC maintenance as well as EMT and may prove to be key targets in eradicating the CSC population and therefore the root of resistance and metastasis [37].Induction of EMT confers re-sistance of NSCLC cells to EGFR-tyrosine kinase inhibitors,however,enforced inhibition of the Hedgehog pathway re-sensitizes the NSCLC cells to EGFR-tyrosine kinase inhibitors through mediation of the EMT process [47].

Previously identi?ed lung CSC subsets have been shown to confer resistance to conventional chemotherapeutics,biological mol-ecules,targeted therapies and radiotherapy used in the current management of lung cancer.The elimination of lung CSCs is of utmost importance at the time of therapeutic intervention in order to prevent CSC expansion and subsequent tumor recurrence,

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Fig.1.Division strategies of cancer stem cells.(A)Cancer stem cells (purple)must accomplish the dual task of self-renewal and generation of differentiated progeny (blue).(B–D)Strategies that maintain a balance of stem cells and differentiated progeny.(B)Symmetric cell division.Each cancer stem cell can give rise to two cancer stem cells or to two differentiated progeny.(C)Asymmetric cell division,each cancer stem cell gives rise to one cancer stem cell and one differentiated cell.(D)Cancer stem cell maintenance may be a combination of symmetric and asymmetric divi-sion strategies.A population strategy provides dynamic control over the balance between stem cells and differentiated progeny,a capacity necessary for the main-tenance or expansion of cancer stem cells.

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and metastasis (Fig.2).Identifying a lung CSC population and elu-cidating its mechanisms of intrinsic resistance may highlight a key molecule or pathway targeting strategy that may prevent tumor re-sistance and recurrence at its source,leading to more effective lung cancer treatments.While little is currently known regarding lung cancer stem cell biology,a number of CSC markers have be iden-ti?ed and studied.These include ALDH1,CD133,side population (Hoechst-negative),CD44,CD87and CD117,all of which have been linked to chemoresistance in a number of ?rst line anti-cancer therapies.

Aldehyde dehydrogenase and resistance

The aldehyde dehydrogenase (ALDH)family members are cy-tosolic isoenzymes responsible for oxidizing intracellular aldehydes,including the oxidation of retinol (vitamin A)to retinoic acid,an important modulator of gene expression and cell differentiation.ALDH is highly expressed in hematopoietic stem cells [49,50].In recent years ALDH1has emerged as a prominent marker for CSCs in a number of solid tumors such as breast,colon,head and neck,prostate [51–55],and lung cancer [56].ALDH1is expressed in a number of NSCLC cell lines,where ALDH1-positive cell subpopu-lations show distinct stem-like characteristics and gene expression pro?les.Low numbers of ALDH1-positive cells,when injected into nude mice have the ability to initiate tumor growth and generate heterogenic tumors.On the other hand,their ALDH1-negative coun-terparts do not demonstrate this ability,indicating that the ALDH1-positive fraction is capable of self-renewal and can give rise to a differentiated cell population.ALDH1-positive cells are highly re-sistant to chemotherapeutic agents commonly used as ?rst-line therapy in the clinical setting,such as cisplatin,gemcitabine,doxo-rubicin,vinorelbine and docetaxel,whereas ALDH1-negative cells are sensitive to the cytotoxic activity of these drugs [56].ALDH1overexpression is associated with poor prognosis in NSCLC pa-tients,where high ALDH1expression is signi?cantly associated with a more aggressive and advanced pathological grade and stage [56,57].Furthermore,increased ALDH1expression has been associated with increased metastasis in multiple cancers [58,59].The Notch pathway is a highly conserved embryogenesis sig-naling pathway involved in the determination of cell fate [60].This pathway has been identi?ed as a key pathway associated with the CSC maintenance of ALDH1-positive lung CSCs.Suppres-sion of Notch signaling with a γ-secretase inhibitor results in a much reduced ALDH1expressing population and decreased pro-liferation and clonogenic capacity of tumor cells [59].In a study by Barr et al.,an isogenic panel of cisplatin resistant NSCLC cell lines was generated by chronic exposure to cisplatin.Increased ALDH1activity correlated with signi?cantly increased expression of CSC and EMT markers,Nanog,Oct-4,Sox-2,c-Met and β-catenin in the cisplatin resistant cell lines relative to their sensitive counterparts [61].

Treatment with paclitaxel has also been shown to induce a greater ALDH1CSC population in H460and H1299lung cancer cell lines,whereas treatment with the selective CSC inhibitor,salinomycin,de-creased the presence of the ALDH1population and reduced the tumorsphere-forming potential of these cells,with no effect on the ALDH1-negative population [62].While paclitaxel treatment in an in vivo model decreased tumor volume,it increased the number of metastatic nodules,most likely due to the expansion of an ALDH1-positive tumor initiating CSC population.Salinomycin alone did not reduce the size of the primary tumor but decreased metastasis [62].In addition to chemotherapy,radiotherapy has also been shown to induce and expand the ALDH1-positive CSC population in lung ad-enocarcinoma A549cells.Repeated 4Gy radiation treatment was used to generate an A549radioresistant cell line,which showed greater proliferative and clonogenic capacity when challenged with radiation,compared to their parental counterparts.Expansion of the ALDH1-positive subpopulation correlated with the development of radioresistance and increased doses of radiation [63].

Resistance to epidermal growth factor receptor (EGFR)tyro-sine kinase inhibitors is a major problem in the treatment of lung cancer.A model of acquired resistance to ge?tinib was estab-lished in a number of EGFR mutated cell lines,EGFR-mutant HCC827(exon19del E746–A750),PC-9(exon19del E746–A750),HCC4006(exon19del L747–E749),and HCC4011(L858R)cells [64].Cell lines were treated using a stepwise dose escalation or high-dose

ge?tinib

Fig.2.Anti-cancer therapies induce CSC expansion.Treatment with conventional chemotherapeutic agents or radiotherapy kills the bulk of tumor cells (blue).However the intrinsically resistant CSCs (purple)survive.Cellular stress caused by chemotherapeutic treatment can induce expansion of these CSCs.The survival and expansion of CSCs promotes tumor relapse and metastasis.

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to generate resistant sublines.Treatment with high-dose ge?tinib induced ALDH1overexpression,increased EMT-associated markers and self-renewal capabilities.These results suggest that acquired resistance is based,at least in part,on the expansion of a stem cell-like population[64,65].Ge?tinib resistant cells exhibit a higher tumor-initiating capability than their parental controls when in-jected subcutaneously into NOD/SCID mice.Ge?tinib resistant sublines displayed resistance to the commonly used chemothera-peutic agents,docetaxel and paclitaxel,an effect that may be due to the expansion of the ALDH1CSC population[64].Isolated ALDH1 cells from ge?tinib resistant PC-9cells showed increased resis-tance to cisplatin,etoposide and?uorouracil compared to ALDH1-negative cells[66].A similar induction of an ALDH1-positive subpopulation was observed during the development of NSCLC re-sistance to the EGFR tyrosine kinase inhibitor,erlotinib.ALDH1-positive cells showed a refractory,inherent resistance to this EGFR TKI.Treatment with a natural polyphenol,silibinin,speci?cally targets erlotinib-refractory ALDH1-positive cells,reducing population size and circumventing erlotinib resistance in vivo,minimizing the ability of the CSCs to evade death and therefore reducing the possibility of recurrence[67].Targeting ALDH1,in addition to one or more CSC surface markers,may offer potential as novel strategies for over-coming the development of treatment resistance in lung cancer (Table1).

CD133and resistance

The CD133antigen,known as prominin1,is a120kDa pentaspan transmembrane protein,encoded by the prom1gene.It is highly expressed on undifferentiated cells,such as hematopoietic stem cells,endothelial progenitor cells and neural stem cells[85–87]. CD133is a well-documented CSC marker that represents a tumor-initiating cell subset in breast,colon,prostate,liver and ovarian solid tumors,as well as SCLC and NSCLC[88–93].Immunohisto-chemical analysis of patient-derived tumors showed variation in CD133positivity among patients,but was not detectable in normal lung tissues[93].SCLC and NSCLC tumors were dissociated and cultured at a low density in serum-free media supplemented with epidermal growth factor and basic?broblast growth factor.Such conditions select undifferentiated CD133-positive cells and promote their enrichment and expansion and induce sphere formation[93,94]. Overexpression of the embryonic stem cell transcription factors Oct-4and Nanog con?rmed the undifferentiated immature phe-notype of CD133-positive cells[93].In the presence of serum,tumor-initiating spheres adhered to the plastic and acquired the morphological features of differentiated cells.CD133expression was lost during differentiation.Subcutaneous injection of lung sphere-forming cells or pure CD133-positive populations into im-munode?cient mice resulted in the growth of tumors with morphological features similar to those seen in the human tumor from which they were derived,con?rming the tumorigenic poten-tial of these CD133expressing sphere-forming cells[93].Injection of differentiated cells or CD133-negative subpopulations using the same animal model resulted in decreased tumorigenic potential and loss of key stemness characteristics.Lung tumor CSC-spheres and CD133-positive cells showed resistance to cisplatin,gemcitabine, paclitaxel,doxorubicin and etoposide when treated with peak plasma concentrations.Resistance was directly correlated with increased expression of the ABC-transporter,ABCG2[93,95].Knock-down of the embryonic cell marker Oct-4speci?cally blocked the ability of CD133-positive cells to form spheres and further promoted their differentiation into CD133-negative cells,thereby reducing the CD133-positive population.Oct-4inhibition decreased the inva-sive and tumorigenic potential of CD133-positive cells in vivo and increased apoptosis of the CD133-positive cells in response to ra-diotherapy in vitro[95].

Platinum-based chemotherapy,such as cisplatin and carboplatin, is an integral?rst-line therapy for patients with NSCLC.However, tumor recurrence and resistance is commonly seen during the course of treatment with these platinum agents.Cisplatin treatment,either acute or chronic,resulted in an8-fold enrichment of CD133-positive fractions.Analysis of CD133in patient tumor resections identi?ed a CD133-positive subtype as a biomarker for predicting shorter progression-free survival[96].This enrichment was thought to be due to drug resistance and recurrence.Low-dose cisplatin treat-ment,su?cient to cause DNA-damage but not cell death,expanded the CD133-positive population and up-regulated ABCG2and ABCB1 drug e?ux-associated proteins in H460and H661cell lines[21]. CD133expansion and stem-cell associated gene expression was de-pendent on cisplatin concentration and exposure time.Cisplatin-primed cells show increased tumor-sphere formation in soft agar colony-forming assays.Cells pre-treated with cisplatin showed in-creased cross-resistance to doxorubicin and paclitaxel.Treatment of side population cells with a speci?c ABCG2inhibitor (pantoprazole)or a pan-inhibitor of ABC transporters(Verapamil) completely abrogated cisplatin-induced resistance[21].

Tumor-hypoxia has long been considered a therapeutic problem in many solid tumors.Recent studies have shown that hypoxic con-ditions signi?cantly increase CD133-positive and Oct-4expressing cells,which correlate with increased ge?tinib resistance.Hypoxic expansion of ge?tinib resistant cells was mediated through the up-regulation of HIF-1αand insulin-like growth factor-1expression and the subsequent activation of insulin-like growth factor-1receptor [97].Inhibition of HIF-1αor insulin-like growth factor-1receptor signi?cantly decreases CD133and Oct-4positive populations and overcomes ge?tinib resistance[97].Elevated CD133expression is associated with intrinsic chemotherapy and radiotherapy resis-tance due to reduced apoptotic response,diminished cell cycle arrest and enhanced DNA repair capacity[98].SCLC chemoresistant CD133-positive cells show increased expression of the mitogenic neuropeptide receptor for gastrin-releasing peptide and arginine va-sopressin.These CD133-positive cells show increased sensitivity to the growth inhibitory and pro-apoptotic effects of broad spec-trum neuropeptide antagonists,highlighting a potential role for neuropeptide antagonists as CSC targeting agents in SCLC[99].

Table1

Potential strategies targeting CSC markers.

CSC Marker Drug Reference

ALDH1Salinomycin[62]

Silibinin[67]

DEAB[68,69]

Disul?ram[70–73]

CD133Pantoprazole[21]

Neuropeptide antagonists[21]

Side population Verapamil[74]

EGFR Inhibitors[75,76]

Obatoclax[77]

GDC-0449[78]

Mithramycin A[79]

Nicardipine[80]

Pravastatin[80]

Secalonic Acid D[81]

Low Weight Molecular

Heparin

[82]

Axitinib[83]

CD117Imatinib[84]

Axitinib[83]

A number of cancer stem cell(CSC)markers identi?ed to date may offer potential

as speci?c targets for therapy,particularly in chemoresistant lung https://www.360docs.net/doc/0d3388140.html,bi-

nation treatments using novel agents or drugs targeting stem-like cells with

conventional anti-cancer treatments may further enhance the e?cacy of chemo-

therapeutic drugs.As such,co-treatment strategies may re-sensitize drug resistant

lung cancer cells to therapy,while at the same time inhibiting expansion of the CSC

population within the tumor.

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A meta-analysis of11studies,consisting of1004NSCLC patient tissues,further con?rmed that high CD133expression correlates with signi?cantly worse5-year overall survival compared to those with low CD133expression.CD133expression was associated with a number of clinical parameters such as tumor stage,grade,differ-entiation status and lymph node metastasis[100,101].

Side population and resistance

The?ow cytometry based side population strategy was initially conceived to enrich or isolate hematopoietic stem cells.It is based on the ability of stem cells to e?ciently e?ux Hoechst33342,and therefore present as a Hoechst-negative population on a dual wave-length?ow cytometry plot.This technique has been used to isolate putative cancer stem cells from a number of solid tumor types,in-cluding prostate,breast,malignant pleural mesothelioma and lung [74,102–105].Tumor side population cells pump out?uorescent Hoechst33342dye(Hoechst-negative),whereas the main bulk pop-ulation,retain the dye(Hoechst-positive).The e?ux capacity of the side population phenotype is determined by ATP-binding cassette membrane transporter G2(ABCG2),also known as breast cancer re-sistance protein1(BCRP1)and is associated with tumor growth, progression and metastasis[106].ABCG2actively e?uxes a wide variety of chemically and structurally unrelated compounds from cells;ABCG2overexpression in tumor cells confers multidrug re-sistance to a multitude of anti-cancer drugs[107].Hoechst staining of NSCLC cell lines con?rmed the presence of a Hoechst-negative side population.However,treatment of cells with verapamil,a calcium-channel blocker and inhibitor of ABCG2,completely elimi-nated the presence of the side population,therefore con?rming the transporter ABCG2as an integral e?ux pump in CSCs.NSCLC side population cells show increased proliferative capacity,tumorige-nicity and overexpression of ABCG2compared to non-side population cells[33,74].

NSCLC side population cells are enriched in the cell cycle G0/G1 phase,have higher ALDH1activity,higher clonogenic survivability and increased self-renewal and differentiation capacities compared to the main cell population.Treatment of NSCLC cell lines(A549,H1650 and H1975)with EGFR tyrosine kinase inhibitors resulted in de-creased side population frequency and ABCG2expression.Downstream regulation of the EGFR-Src-Akt signaling axis resulted in signi?cant inhibition of Sox-2.This inhibition resulted in the abrogation of the self-renewal and metastatic abilities of the side population cells[75,76].β-arrestin is involved in the progression of NSCLC,where its inhibi-tion has been shown to signi?cantly decrease the presence of the side population and inhibit the growth and expansion of A549cells.The Bcl-2family member,MCL-1,is overexpressed in the side popula-tion and is associated with the self-renewal of CSCs.MCL-1speci?c inhibition,with Obatoclax,prevented self-renewal of EGFR–resistant NSCLC cells[77].The Hedgehog pathway has long been implicated in the maintenance of CSCs.Treatment of adenocarcinoma cells (HCC)and SCLC cells(H1339)with the Hedgehog inhibitor GDC-0449,inhibited cell growth and signi?cantly decreased side population frequency.Hedgehog inhibition in combination with cisplatin in-creased the cytotoxic effects of cisplatin when compared to cisplatin-only treatment[78].Small molecule inhibition or gene silencing of CK2αresulted in the down-regulation of the Hedgehog pathway and subsequently induced a reduction of the side population percent-age[108].

Cigarette smoking at diagnosis or during lung cancer treat-ment is associated with poor prognosis and cigarette smoke has now been shown to promote drug resistance via the expansion of the CSC side population[109].Treatment of A549adenocarcinoma cells with cigarette smoke condensate resulted in a signi?cant increase in side population frequency,ABCG2expression and doxorubicin e?ux.This effect was mediated by PI3K/Akt signaling.PI3K inhi-bition and subsequent ABCG2down-regulation and decreased side population occurrence has been shown to re-sensitize adenocar-cinoma(A549)and malignant pleural mesothelioma(ZL55and SDM103T2)cell lines to doxorubicin and mitoxantrane,respective-ly[109,110].In a similar study,treatment of A549and Calu-6cell lines with mithramycin A(a selective inhibitor of the binding of tran-scription factor Sp1to the ABCG2promoter region)repressed smoke-induced expression of ABCG2and side population frequency[79,111].

Occurrence of the side population has been associated with re-sistance to many chemotherapeutic agents and targeted therapies used today due to its characteristic excessive e?ux capacity.Tar-geting side population cells may prove to be an important mechanism to overcome resistance to a number of therapies by reducing drug e?ux from cells,thereby increasing drug retention within the cells. In a recent study by Wu et al.,NSCLC patients exhibiting chemo-resistance to platinum-based chemotherapy had elevated serum levels of cholesterol and increased expression of ABCG2[80].In an in vitro model of NSCLC using A549cells,cholesterol increased che-moresistance while inhibition of ABCG2with Nicardipine reversed cholesterol-induced platinum-resistance.Similarly,treatment with the statin Pravastatin,re-sensitized A549cells to cisplatin,carboplatin and oxaliplatin.This study suggests that lung cancer patients with high cholesterol may respond to platinum-based therapy when com-bined with a statin[80].Secalonic acid D,a marine fungal-derived metabolite,has a potent anti-cancer effect through its inhibition of ABCB1,ABCC1and most signi?cantly ABCG2,in multidrug resis-tant cells.Treatment with Secalonic acid D signi?cantly increased ABCG2degradation via the activation of caplain1,decreased the percentage of side population cells and inhibited the sphere-forming abilities of the side population CSC cells[81].Low weight molecular heparin has been suggested to affect the biological prop-erties of side population cells.Treatment of side population cells with low weight molecular heparin decreased the sphere-forming abilities of the CSC population through the degradation of ABCG2. While treatment of the cisplatin resistant side population cells with low weight molecular heparin alone did not affect their prolifera-tive capacity,the side population cells were re-sensitized to cisplatin-induced apoptosis when used in combination with cisplatin[82]. CD44and resistance

CD44is an integral membrane glycoprotein that functions as a receptor for the extracellular matrix glycan,hyaluronan.CD44is ex-pressed on the surface of speci?c glandular,?broblastoid and hematopoietic cells[112].CD44has been identi?ed as an impor-tant marker of CSCs in breast,prostate,pancreatic,squamous head and neck,and in more recent years,lung cancer[113–116].NSCLC cells expressing CD44are enriched for stem-like properties.CD44-positive cells showed increased spheroid body formation,enhanced self-renewal abilities and increased tumor initiating capacity in vivo compared to their negative counterparts,and the heterogenic CD44 population from which they were derived[117].CD44-positive cells showed increased expression of pluripotency markers(Nanog,Oct-4,Sox-2)and EMT markers(SNAI1,CDH2and VIM)and increased resistance to cisplatin[117].NSCLC cells surviving5Gy radiation therapy were enriched for CD44expressing cells,and showed in-creased tumor initiating potential,indicating an inherent radioresistance within the CD44-positive subpopulation[118].Both lung cancer and malignant pleural mesothelioma cells co-expressing CD44with ALDH1showed increased drug resistance,sphere-forming capacity and tumorigenicity[15,68].

Immunohistochemical staining of159resected NSCLC tumors showed that CD44expression is more frequently detected in squa-mous cell carcinoma compared to adenocarcinoma histology.High CD44expression was also signi?cantly correlated with advanced regional lymph node metastasis and is a signi?cant predictor of a more advanced tumor status in squamous cell carcinomas[119].

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CD87and resistance

Numerous solid tumors,including lung cancer,show increased expression of urokinase plasminogen activator(uPA)and its recep-tor,urokinase plasminogen activator receptor(uPAR),otherwise known as CD87.CD87is highly involved in migration and in the reg-ulation of cell adhesion.High expression of CD87is strongly correlated with an unfavorable clinical outcome and a signi?-cantly shorter overall survival in SCLC[120,121].In an in vivo model of NSCLC,simultaneous inhibition of CD87and MMP-9inhibited tumor growth,invasion,metastasis and angiogenesis[122].Reduc-tion of CD87signaling increased cisplatin-induced apoptosis and anoikis,a method of programmed cell death induced by anchorage-dependent cells detaching from the extracellular matrix[123].Gutova et al.identi?ed a rare population of CD87-positive cells in a panel of human SCLC cell lines(H1688,H1417,H69AR,H211,H1882and H250).Isolation of these CD87-positive cells con?rmed the expres-sion of stemness and CSC markers,such as CD44and MDR1, respectively.The CD87-positive population demonstrated high sphere-forming capabilities,increased tumor initiating potential,as well as signi?cant resistance to numerous traditional chemothera-peutic agents,including cisplatin,etoposide and paclitaxel [120,124,125].

CD117and resistance

CD117(KIT,c-KIT)is a type III receptor tyrosine kinase that is phosphorylated upon binding of its ligand,stem cell factor(SCF), leading to a signaling cascade that culminates in the alteration of a number of biological processes,such as apoptosis,cell differen-tiation,cell adhesion and proliferation[126].SCF and CD117are overexpressed in a number of human malignancies,including gas-trointestinal stromal tumors(GIST)[127],breast cancer[128],acute myeloid leukaemia(AML)[129]and lung cancer[130].High SCF ex-pression in lung adenocarcinoma is strongly associated with smoking status and poor prognosis[131].Overexpression of CD117within lung tumors is also associated with poor prognosis,lower survival rates and chemoresistance[84].NSCLC cell lines,H460and A549, when grown in CSC selective media,gave rise to tumorspheres that exhibit increased expression of CD133,CD117,Oct-4,TRA-1-81,in-creased nuclear expression ofβ-catenin and resistance to chemotherapy[84].CSC-spheres also showed high expression of SCF, whereas SCF was not detectable in the bulk cell population.Exog-enous SCF expression increased CSC-sphere size and stimulated their proliferation.Treatment of the CD117-expressing CSC-spheres with Imatinib(Gleevec),an FDA-approved tyrosine kinase CD117inhib-itor commonly used in the treatment of AML and GIST,dramatically reduced CSC-sphere size and number.Imatinib given in combina-tion with cisplatin or doxorubicin led to a potent elimination of both CSC-sphere forming cells and the bulk cell population[84]. Targeting CSCs to overcome therapeutic resistance in lung cancer

Speci?c targeting of cancer stem cells in combination with?rst-line chemotherapeutic agents holds great promise as a strategy to overcome chemoresistance,tumor relapse and metastasis(Fig.3). However,such speci?c targeting of CSCs has not been extensively explored as a therapeutic option in the treatment of lung cancer. Inhibition of CSC markers,such as ABCG2,has been shown to enhance the e?cacy of chemotherapeutic agents in CSC cells[83]. Axitinib is an oral,potent,small molecule ATP competitive multi-target tyrosine kinase inhibitor.It inhibits CSC markers CD117and ABCG2.Axitinib has also been shown to reverse multidrug resis-tance via ABCG2inhibition both in vitro and in vivo.Axitinib–doxorubicin combination treatment promoted intracellular accumulation of doxorubicin within side population CSC cells,and signi?cantly enhanced the cytotoxic effects of doxorubicin[83].

An increasing body of evidence is beginning to emerge in rela-tion to the inhibition of ALDH1in stem-like tumor cells that are resistant to therapy[132].Conventional chemotherapy has had little effect in the treatment of malignant pleural mesothelioma.However, combination treatment of ALDH-high,CD44-positive subpopula-tions isolated from three mesothelioma cell lines(H28,H2052and Meso4)with the ALDH1inhibitor,diethylaminobenzaldehyde(DEAB), increased cisplatin sensitivity and signi?cantly reduced cell viabil-ity[68].Knockdown(siRNA)or chemical inhibition(using DEAB

Fig.3.Future implications for CSC-targeted therapies.(A)Current treatment strategies focus on debulking the tumor(blue)but not the intrinsically resistant CSC subset (purple).Chemotherapy kills the majority of cells within the main tumor cell population.However the CSC subset survives and gives rise to new,therapeutically resistant heterogenic tumors and metastases.(B)Combining current cytotoxic therapies with novel CSC-targeted therapies may eradicate this CSC subset and therefore inhibit the root of chemoresistance,tumor recurrence and metastasis,thereby improving clinical outcomes.

152L.MacDonagh et al./Cancer Letters372(2016)147–156

disul?ram)of ALDH1A1in A549and H522lung cancer cells de-creased the cells proliferative and migratory capabilities[69].Of interest is the recent re-purposing of the FDA-approved pan-ALDH1inhibitor,disul?ram(Antabuse),originally used in the treatment of chronic alcoholism[133].In more recent years,a number of studies have demonstrated the potential use of disul?-ram in targeting ALDH1expressing CSCs in breast,cervical,prostate, melanoma and lung tumors[134–138].As such,disul?ram was used to examine its effects in re-sensitizing cells to current therapies or enhancing the cytotoxic effects of platinum-based chemotherapy and radiation therapy[70,71].When disul?ram is administered in combination with copper chloride in vitro,it acts as an effective proteasome inhibitor and as a copper ionophore.Disul?ram–copper complexes are formed and induce copper-dependent oxidative stress,exerting its potent apoptotic and anti-cancer effects [72].In a small phase IIb trial,disul?ram in combination with cisplatin and vinorelbine was well-tolerated and signi?cantly pro-longed overall patient survival in metastatic NSCLC patients[73]. Interestingly,of the forty patients included in the trial,there were two long-term survivors,both of whom were in the disul?ram com-bination treatment group.

The presence of hyper-malignant or CSCs in lung cancer and other tumor types are largely responsible for the failure of current anti-cancer treatments with metastasis and relapse accounting for a high percentage of patients.In a recent study by Li et al.,the small mol-ecule inhibitor BBI608inhibited gene transcription driven by the signaling transducer and activator of transcription3(STAT3)[139]. BBI608abrogated cancer stem cell properties including tumorsphere formation and self-renewal capabilities through the depletion of cancer stem cells.BBI608treatment effectively blocks cancer relapse and metastasis in mice and holds great promise as a future anti-stemness therapy[139].

Conclusion

Despite the development of targeted therapies,there has been little improvement in the prognosis of lung cancer patients.The cancer stem cell hypothesis has provided a new avenue of focus in overcoming resistance to therapy,preventing tumor relapse and im-proving progression-free and overall survival rates in lung cancer patients.Signaling pathways associated with stem cell properties, such as differentiation and self-renewal capacities in addition to the identi?cation of CSC markers,offer potential targets for novel anti-cancer strategies.While a number of pre-clinical and clinical studies (Table2)examining CSC-targeted agents are still in their infancy,many have been shown to be well tolerated and show enhanced e?cacy when used in combination with conventional cytotoxic chemotherapies.

Acknowledgments

This work was funded by Molecular Medicine Ireland(MMI),as part of the Clinical&Translational Research Scholars Programme (CTRSP).

Con?ict of interest

The authors have no con?ict of interest to declare. References

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Target Agent Phase Clinical Trials

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NCT02259582 STAT3Methformin II NCT01717482 STAT3BBI608I/II NCT02347917

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NCT02084823 Sp1Mithramycin A II NCT01624090 ALDH1Disul?ram II/III NCT00312819

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