Dittmer-2018-Breast cancer stem cells_ Feature.pdf

Contents lists available at ScienceDirectSeminars in Cancer Biologyjournal homepage: cancer stem cells:Features,key drivers and treatment optionsJrgen DittmerClinic for Gynecology,Martin Luther University Halle-Wittenberg,GermanyA R T I C L E I N F OKeywords:Breast cancerCancer stem cellsNotchWntHedgehogOSKMA B S T R A C TThe current view is that breast cancer is a stem cell disease characterized by the existence of cancer cells withstem-like features and tumor-initiating potential.These cells are made responsible for tumor dissemination andmetastasis.Common therapies by chemotherapeutic drugs fail to eradicate these cells and rather increase thepool of cancer stem cells in tumors,an effect that may increase the likelyhood of recurrence.Fifteen years afterthe first evidence for a small stem-like subpopulation playing a major role in breast cancer initiation has beenpublished a large body of knowledge has been accumulated regarding the signaling cascades and proteins in-volved in maintaining stemness in breast cancer.Differences in the stem cell pool size and in mechanismsregulating stemness in the different breast cancer subtypes have emerged.Overall,this knowledge offers newapproaches to intervene with breast cancer stem cell activity.New options are particularly needed for thetreatment of triple-negative breast cancer subtype,which is particularly rich in cancer stem cells and is also thesubtype for which specific therapies are still not available.1.IntroductionBreast cancer is the most frequent cancer among women worldwideand overall the leading cause of cancer-related death in women 1.Breast cancer is not a homogenous disease.Different subgroups can bedistinguished by immunohistochemistry and by mRNA analysis.Im-munohistochemical analysis are commonly performed to determine thestatus of ER(estrogen receptor),PR(progesterone receptor),andHer2(human epidermal receptor 2).They serve as biomarkers fortreatment.ER-positive tumors are responsive to anti-estrogens andaromatase inhibitors,which both interfere with ER function 2,Her2-positive tumors react to Her2-directed drugs,such as the anti-Her2antibody trastuzumab 3.Approximately two third of all breast can-cers express ER and 15%Her2 at levels that justify target-specifictreatment.A third immunohistochemical subtype,TNBC(triple-nega-tive breast cancer),is devoid of ER,PR and Her2 and represents15%of all breast cancers.There is currently no target-specifictherapy available for TNBCs.Instead TNBC patients are treated bychemotherapy,most often in a neoadjuvant fashion 4.Chemotherapyis also commonly used in advanced ER-positive and in Her2-positivetumors.Generally,irradiation is often applied as an additional treat-ment in breast cancer 5.The improvement of therapies has con-tributed to the decline in breast cancer mortality in developed countries6,7.RNA analysis has revealed further heterogeneity among breastcancers.Five major subgroups could be distinguished based on RNAexpression signatures:luminal A,luminal B,Her2-enriched,claudin-low,basal-like 8.These molecular(intrinsic)subtypes overlap withthe immunohistochemical subtypes.Most luminal A and B are ER-positive,most Her2-enriched are Her2-positive,and most basal-like areTNBCs.The differentiation of ER-positive in luminal A and luminal Bhas proved to be of importance for treatment.Luminal B cancers aregenerally more aggressive than luminal A cancers and show oftenhigher expression of the proliferation marker Ki67 and/or express Her2along with ER 9.The basal-like subtype represents not all TNBCs.Additional molecular subtypes of TNBCs are the mesenchymal-likesubtype,which include also claudin-low cancers and the luminal an-drogen receptor subtype 10.The different TNBC subtypes are asso-ciated with different outcomes.Breast cancer is a systemic disease characterized by early tumor celldissemination 11.Disseminated tumor cells show high resistance tosystemic therapies and can give rise to lethal metastasic lesions 12.Metastatic dormancy delays the formation of macrometastasis 13 andit may take years before a fatal metastastic disease has been developed14.The current view is that most solid cancers,including breast cancer,are stem cell diseases 15,16 and that these stem cells are critical fordissemination and metastasis 17,18.The groundwork for the conceptof stem-like cells being involved in the initiation of breast cancer1921 was laid by data reported by Al-Hajj and colleagues 22.Theyhttps:/doi.org/10.1016/j.semcancer.2018.07.007Received 18 May 2018;Received in revised form 10 July 2018;Accepted 18 July 2018Correspondence to:Klinik fr Gynkologie,Universitt Halle,Ernst-Grube-Str.40,06120,Halle/Saale,Germany.E-mail address:juergen.dittmermedizin.uni-halle.de.Seminars in Cancer Biology 53(2018)5974Available online 27 July 20181044-579X/2018 Elsevier Ltd.All rights reserved.Tshowed that only a small subpopulation of breast cancer cells have thepotential to form tumors in mice.These tumor-initiating cells werefound to be able to rebuild the tumor with a similar heterogeneity asfound with the original tumor suggesting that these cells show plasticityreminescent of stem cells 22.The term“cancer stem cells(CSCs)”wasborn to define tumor-initiating cells with the capability of self-renewaland“the ability to recapitulate the generation of a continously growingtumor”19.Mammosphere formation assays allowing the identicationof functional CSCs in vitro and FACS analysis to identify CSCs as sidepopulations facilitated research on CSCs 23.This review aims to give an overview of the current knowledge onbreast cancer stem cells in terms of their particular features,the keydrivers of their maintenance and options to specifically target anderadicate them.2.Features of breast cancer stem cells2.1.CD44+/CD24/lowand ALDEFLOUR-positive breast cancer stem cellsBiochemically breast CSCs(BCSCs)were originally characterized bythe expression of the surface marker CD44 and the lack or low ex-pression of the surface marker CD24 22,24.CD44-and CD24-positivecancer cells isolated from breast cancer specimens show different genesignatures with the signature linked to CD44+cells being associatedwith a significantly worse prognosis 24.Among others,the CD44+/CD24/lowsubpopulations express PROCR(protein C receptor)athigher levels 24,25,a factor important for the development andmaintenance of the mammary gland 26.CD44,a hyaluronan receptor,is involved in many biological activities of tumor cells,such as migra-tion and extravasation 27.CD44 was also found to be a key marker ofcirculating tumor cells that are able to initiate metastasis 17.Theexistence of many splicing variants suggests that CD44 biology iscomplex.In support of this notion,the different breast cancer subtypesshow different preferences for certain CD44 isoforms 28.Breastcancer subtypes also differ in the proportion of the CD44+/CD24/lowCSC subpopulation.In basal-like tumors,this proportion is higher thanin luminal A and B tumors,more often reaching or exceeding 10%asdetermined by immunohistochemistry 29.The tumor-initiating potential of CD44+/CD24/low-BCSCs ishigher,if they are positive for EpCAM(epithelial cell adhesion mole-cule),a transmembrane glycoprotein also called epithelial-specific an-tigen(ESA)22.Prediction of the tumor-initiating potentials of humanbreast cancer cell lines was more accurate when based on the number ofEpCAM-positiveCD44+/CD24/lowBCSCs30confirmingthatEpCAM is important for tumor initiation.Furthermore,EpCAM ex-pression in murine 4T1 breast cancer cells has been linked to self-re-newal and enhanced bone metastasis formation 31.One way bywhich EpCAM may affect CSC activity is by supporting the activity ofthe Wnt pathway 32,a pathway involved in CSC maintenance(seeSection 3.2.).EpCAM plays also a role in more differentiated epithelial-like breast cancer cells.In these cells,which express EpCAM at higherlevels than BCSCs,EpCAM is involved in the regulation of proliferationand adhesion 32.Interestingly,EpCAM expression helps to distin-guish different types of cells(luminal non-CSCs,CSCs and basal non-CSCs)in basal-like breast cancer cell lines 33.EpCAM expression ishighest in luminal non-CSCs(CD44low/CD24high),lower in CSCs(CD44high/CD24-)and not detectable in the basal non-CSCs(CD44high/CD24-).A different type of BCSC was found when breast cancer cells weresorted based on their ALDH(aldehyde dehydrogenase)activity by usingan ALDEFLOUR assay 34.The ALDEFLOUR-positive CSC populationonly partially overlaps with the CD44+/CD24/lowpopulation.Bipo-tent ALDEFLOUR-positive CD44+/CD24/low-CSCs show the highestBCSC activity 34,35.ALDHs are engaged in aldehyde detoxification36.Of the 19 ALDH members,four(ALDH1 A1,ALDH1 A2,ALDH1A3 and ALDH8A1)may be of particular importance for CSC activity,asthey interfere with retinoic acid signaling 37,which negatively in-terferes with BCSC activity 38.An association between high expres-sion and poor prognosis has been reported for ALDH1A1 and ALDH3A13941.Also,high anti-ALDH1 immunoreactivity in ER-and Her2-positive tumor samples was found to be linked to early recurrence andmore aggressive disease 42.Furthermore,ALDH1A1 has been re-ported to regulate branching during mammopoiesis by interfering withretinoic acid activity 43.DetailedanalysisoftheCD44+/CD24/lowandALDEFL-OUR-positive subpopulations revealed that CD44+/CD24/low-BCSCsdisplay a mesenchymal and ALDEFLOUR-positive BCSCs an epithelialphenotype and that BCSCs can reversibly transit between these twostates 44,processes called EMT(epithelial-to-mesenchymal transi-tion)and MET(mesenchymal-to-epithelial transition)(see Section2.2.).The mesenchymal BCSCs(EMT-BCSCs)were found to be moreinvasive,but quiescent,whereas the epithelial BCSCs(MET-BCSCs)areless invasive,but mitotically active.The bipotent ALDEFLOUR-positiveCD44+/CD24/lowBCSC is supposedly the transit form between thesetwo states 45.It has been suggested that differentiated epithelialbreast cancer cells emerge from epithelial BCSCs and differentiatedmesenchymal cells from mesenchymal BCSCs 46.This may explainwhy luminal breast cancer with a higher proportion of ALDEFL-OUR-positive MET-BCSCs show an epithelial-like phenotype,whereasclaudin-low breast cancer with a higher proportion of CD44+/CD24/lowEMT-BCSCs display a mesenchymal phenotype 45.2.2.The epithelial-to-mesenchymal transition and stemnessEMT and its reverse process,MET,are well-studied phenomenonswhich are essential for embryonic development,wound healing andcarcinogenesis 47.The transition to a mesenchymal phenotype be-stows cells with migratory and invasive capabilities.In invasive ductalbreast cancer,the majority of circulating tumor cells(CTCs)displaymesenchymal characteristics independent of the breast cancer subtypethey originated from 48,49.Hence,it has been suggested that pri-marily cancer cells with mesenchymal phenotype enter the bloodstream and disseminate to remote organs 18,50.Since metastatic le-sions often show an epithelial-like appearance,it is further proposedthat the mesenchymal cells undergo MET at the metastatic site.Thismodel postulates that CTCs have metastatic activity and that EMT islinked to metastasis 51,52.Both hypotheses are supported by ex-perimental evidence 17,53,54.Recently,it has been shown that a 16-gene signature,that include 13 important mesenchymal markers,islinked to poor prognosis in a pan-cancer analysis 55 confirming theimportance of EMT in cancer progression.Importantly,EMT can bestow cells also with stem cell properties.Simply the loss of E-cadherin is able to convert epithelial-like breastcancer cells to stem-like cells that share many characteristics with theCD44+/CD24/low-BCSCs 56.EMT is induced by activation of EMTtranscription factors(EMT-TFs),such as Twist,ZEB1,Snail,Slug andFOXC2(forkhead box C1)51.The major functions of EMT-TFs is todownregulate genes involved in cell adhesion,such as E-cadherin,undto upregulate those responsible for migration and invasion 18.Alongwith the loss of E-cadherin,the Wnt pathway effector-catenin(seeSection 3.2.)is released from its complex with E-cadherin and con-tributes to the acquisition of stem-like features 57.One of the keyfactors that induce the activation of EMT-TFs is TGF(transforminggrowth factor)58,59.TGF has been shown to cooperate with theWnt pathway to induce EMT 60.CD44+/CD24/low-CSCs isolatedfrom breast cancer specimens show increased activities of both theTGF and Wnt signaling pathways 24,25.In the mesenchymal TNBCline MDA-MB-231,expression of YAP1(Yes-associated protein 1),acomponent of the Hippo pathway 61,has recently been found topromote the generation of CD44+/CD24/low-CSC population,whilereducing the fraction of ALDEFLOUR-positive CSC population 62.YAP1 is able to induce EMT in tumors 63 and may therefore supportJ.DittmerSeminars in Cancer Biology 53(2018)597460the mesenchymal status of BCSCs.Similarly,the YAP relative TAZ(transcriptional coactivator with PDZ-binding motif)has been shown toincrease the CD44+/CD24/low-CSC population 64.Also,annexin A3fosters the formation of the CD44+/CD24/low-BCSC at the expense ofALDEFLOUR-positive CSC subpopulation in the MDA-MB-231 andMDA-MB-468 cell lines 65.Annexin A3 was found to upregulate theNFB(nuclear factor B)pathway,a pro-survival pathway involved inself-renewal of BCSCs 66,by inhibiting its inhibitor IB 65.2.3.Drug-and radioresistance of breast CSCsA prominent feature of CSCs is their lower sensitivity to drugs andirradiation than non-CSCs 93.This is the main reason why followingtreatment with commonly used drugs the proportion of the BCSC po-pulation often raises(Table 1),whereby this rise may also be caused byrecruitment of BCSCs from the non-BCSC population 71,94,95.Theacquisition of drug resistance has also been shown to be associated witha higher proportion of the BCSC population 96.In addition,TNBCpatients with no pCR(pathologic complete response),meaning thatresidual tumor is still left after neoadjuvant treatment,exhibit moreALDH1-positive cells in pre-treatment samples than patient with pCR97.A number of chemotherapeutic drugs have been shown to foster CSCactivity.For instance,paclitaxel,epirubicin and cisplatin are able toinduce an elevation of the ALDEFLOUR-positive and/or CD44+/CD24-CSC populations relative to the non-CSC population in MDA-MB-231and MCF-7 cell lines 67,70,81,82,87.Likewise,doxorubicin,cisplatin,5-FU(5-fluorouracil)or a combination of drugs(5-FU,doxorubicin andcyclophosphamide)upregulated the proportion of the CD44+/CD24-CSC population in the T47D cell line 71,7577.Along with thetreatment-induced promotion of the CSC population the tumor-in-itiating potential of breast cancer cells rises.This was shown in vitro byusing mammosphere assays 72,75,81,87,88 as well as in vivo by xe-nograft experiments 67,82,88,89.Hence,chemotherapy-induced CSCpromotion may facilitate recurrence.Consistent with this assumption,Arnold et al.reported that,shortly after the tumor volume was reducedby docetaxel,ALDH1A3 expr