Stem cells in cancer: instigators and propagators? · Commentary 2357 Introduction Cancer begins in...

Commentary 2357

IntroductionCancer begins in normal somatic cells with a mutation that isfixed by a round of cell proliferation – a process termed initiation.If the mutated cell has a selective growth advantage over itsnormal cell neighbours owing to, for example, enhancedproliferation or resistance to apoptosis, then a clone of cellscarrying the same mutation will emerge; in the colon this wouldmanifest as a small adenoma. Further tumour progression can beviewed as an evolutionary process: if a second mutation in onecell of the nascent cell clone gives that particular cell a furthergrowth advantage, then a new clone will outgrow the precedingclone; in the colon, this would manifest as a larger adenoma withmore cellular irregularity (dysplasia). Further rounds of mutationand clonal expansion give rise to progressively more abnormalclones until an additional mutation produces the malignant cellphenotype that can invade beyond normal tissue boundaries and,subsequently, spread to distant sites – a phenomenon known asmetastasis.

We now believe that most tumours contain a subpopulation ofmalignant cells with stem cell properties. These stem cells can giverise to new tumours when xenografted, usually in small numbers,into immunodeficient mice – hence, we refer to them as tumour-propagating cells (TPCs). However, it is currently unclear whetherTPCs arise directly from the transformation of normal stem cellsor whether they are derived from aberrant de-differentiation ofmore mature neoplastic cells at a later stage of cancer progression.

Microscopic inspection of most tumours reveals a complexheterogeneous picture. Tumours have a hierarchical nature, in partbecause TPCs can give rise to more TPCs as well as to transit-amplifying cells (TACs) and terminally differentiated (TD) cells(Fig. 1). However, phenotypic and behavioural heterogeneity isalso generated by variation within different parts of a tumour withrespect to their proximity to the vascular network, and because notall of the cells are cancer cells. Non-cancer cells within a tumourcan include inflammatory cells, cancer-associated fibroblasts and

immature myeloid cells, all of which influence tumour behaviourand often facilitate invasion and metastasis (Coussens and Werb,2002; Alison et al., 2009b). The microenvironment surroundingblood vessels is conducive to the highest rates of tumour-cellproliferation, but it can also serve as the local stem cell niche, aspecialised microenvironment of nerves, mesenchymal cells andextracellular matrix molecules that regulate aspects of stem cellbehaviour, particularly the choice between asymmetric division(which underlies self-renewal) and symmetric division (whichleads to an increase in the number of stem cells) (Alison and Islam,2009).

In this Commentary, we discuss various lines of evidence thatillustrate how stem cells are central to cancer biology. First,mutation in the DNA of normal stem cells appears to be theinitiating event in many (although not all) types of cancer. Second,cancers themselves have stem cells (referred to here as TPCs).Third, TPCs can be prospectively isolated based on the expressionof specific surface markers or intracellular enzyme activity. Fourth,TPCs can be remarkably resistant to therapies that kill the majorityof cancer cells. Fifth, there are several new and, hopefully, more-effective therapies currently being designed to target the uniqueattributes of these cells.

Normal stem and progenitor cells and theorigins of cancerIt is widely believed that tumorigenesis commonly begins in normaladult stem cells or progenitor cells that have recently descendedfrom them. This concept is supported first by the fact that stemcells have – or can re-acquire – the self-renewal mechanismsneeded for maintaining and expanding stem cell numbers. Second,stem cells are anchored to the niche and are therefore not sweptaway in the prevailing cell flux, allowing time to acquire furthermutations (Fig. 1); this is particularly pertinent to cancers ofcontinually renewing populations, such as cells of thehaematopoietic system, gut and skin.

Stem cells in cancer: instigators and propagators?Malcolm R. Alison1,*, Shahriar Islam1 and Nicholas A. Wright2

1Centre for Diabetes, and 2Centre for Digestive Diseases, Blizard Institute of Cell and Molecular Science, Barts and The London School ofMedicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK*Author for correspondence ([email protected])

Journal of Cell Science 123, 2357-2368 © 2010. Published by The Company of Biologists Ltddoi:10.1242/jcs.054296

SummaryThere is growing realization that many – if not all – cancer-cell populations contain a subpopulation of self-renewing stem cells knownas cancer stem cells (CSCs). Unlike normal adult stem cells that remain constant in number, CSCs can increase in number as tumoursgrow, and give rise to progeny that can be both locally invasive and colonise distant sites – the two hallmarks of malignancy.Immunodeficient mouse models in which human tumours can be xenografted provide persuasive evidence that CSCs are present inhuman leukaemias and many types of solid tumour. In addition, many studies have found similar subpopulations in mouse tumoursthat show enhanced tumorigenic properties when they are transplanted into histocompatible mice. In this Commentary, we refer toCSCs as tumour-propagating cells (TPCs), a term that reflects the assays that are currently employed to identify them. We first discussevidence that cancer can originate from normal stem cells or closely related descendants. We then outline the attributes of TPCs andreview studies in which they have been identified in various cancers. Finally, we discuss the implications of these findings forsuccessful cancer therapies.

Key words: Clonogenicity, Immunodeficient mice, Self-renewal, Stem cells, Tumour-propagating cells

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Small intestineIn the mouse small intestine there is both a slowly cycling stemcell population found ~4-5 cell positions above the base of thecrypt (Sangiorgi and Capecchi, 2008), and a rapidly cycling stemcell population composed of slender-shaped cells [so-called cryptbase columnar cells (CBCCs)] that are sandwiched between Panethcells at the base of the crypt (Barker et al., 2007). The stem cellcharacteristics of the two compartments have been establishedusing elegant genetic-lineage tracking techniques. The former cellpopulation expresses Bmi1, a component of the polycomb-repressing complex 1 (PRC1) that prevents stem cell senescence;this Bmi1-expressing population can give rise to clones that containall intestinal lineages. CBCCs, by contrast, express the Wnt targetgene Lgr5, which encodes an orphan G-protein-coupled receptor(Barker and Clevers, 2010); these Lgr5-expressing cells are alsomultipotential stem cells (Barker et al., 2007). Targeted deletion ofthe tumour suppressor gene Apc (adenomatous polyposis coli) inLgr5-expressing CBCCs resulted in rapidly growing adenomas,whereas targeted deletion of Apc in the higher-positioned TACsthat are the direct descendants of either stem cell population failedto induce substantial adenoma growth (Barker et al., 2009). Thesedata illustrate that a mutation in a stem cell population is mosteffective for tumour initiation in the short term. However, smalladenomas derived from TACs persisted for nearly a year in thismodel, so the possibility that cancer later arises from theseadenomas cannot be excluded, particularly given the fact thathuman colorectal cancers grow very slowly (Camplejohn et al.,1973). Intriguingly, 36 days after induction of the stem-cell-derivedadenomas, both colonic and small intestinal adenomas contained~6.5% Lgr5-positive (Lgr5+) cells; it would be interesting to knowwhether these cells become TPCs. The expression of Lgr5 in micecan occur in the same cells that express prominin 1 (PROM1, alsoknown as CD133, a family member of glycosylated pentaspanmembrane proteins; Box 1). Activation of Wnt signallingspecifically in these CD133-positive (CD133+) cells demonstratedthat all neoplastic tissue came from CD133+ cells (Zhu et al., 2009).Remarkably, the frequency of CD133+ cells in the adenomas (7%)was the same as the frequency of Lgr5+ cells in the adenomasgenerated in the study in which Apc was deleted from the Lgr5-expressing population. Intestinal tumours may also be initiatedfrom the stem cell population at positions 4-5 above the base ofthe crypt. These cells overexpress Wip1 phosphatase, an enzymethat turns off the DNA-damage response through inactivation ofstress-induced kinases such as Chk2. ApcMin/+ mice are a modelof familial adenomatous polyposis; the absence of Wip1 in ApcMin/+

Wip1-null mice severely reduces adenoma burden through p53-dependent apoptosis of damaged stem cells because the DNA-damage response is extended (Demidov et al., 2007). The absenceof the hyaluronan receptor CD44 also reduces adenoma burden inApcMin/+ mice, probably because of increased apoptosis of initiatedcells. As CD44 is expressed only by CBCCs and early TACs inintestinal tissues, this further supports the idea that these cells caninitiate adenomas (Zeilstra et al., 2008).

ProstateIt was previously thought that all tumours in the human prostategland arose from androgen receptor (AR)-negative (AR–) basalstem cells (Maitland and Collins, 2008). Indeed, androgen inhibitiondoes not reduce the clonogenicity of prostatic cancer cell linesbut does reduce the size of individual colonies, consistent with thefinding that androgens are unnecessary for the maintenance of

stem cells but required for proliferation of TACs (Bisson andProwse, 2009). However, in some human prostate cancer cell lines,CD133+ TPCs are also AR-positive (AR+), suggesting that theyoriginated from differentiated luminal cells (Vander-Griend et al.,2008). Likewise, in mice, a rare population (<1% of total) ofcastration-resistant luminal cells that expresses the transcriptionfactor Nkx3-1 (a negative regulator of prostatic epithelialproliferation) was found to have self-renewing and bipotentialproperties. Furthermore, targeted deletion of the phosphatase Pten

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Box 1. Defining markers of human TPCs

ALDHThe ALDH gene superfamily encodes detoxifying enzymes; highexpression of ALDH can be detrimental to tumour eradication (Dylla etal., 2008).

Bmi1Polycomb protein; suppresses the INK4A and ARF genes.

CD24A sialoglycoprotein that acts as a ligand for P-selectin. It enables cellsto bind to platelets – tumour-platelet thrombi protect cells in thebloodstream and in turn facilitate tumour invasion through interactingwith endothelia.

CD44A transmembrane glycoprotein that binds hyaluronan (HA); oftenexpressed in various isoforms.

CD90Also known as Thy-1; a glycosylphosphatidylinositol-anchored protein.

CD105A type I integral membrane protein.

CD117Also known as c-kit; a tyrosine kinase receptor for SCF.

CD133The first identified member of the prominin family of pentaspanmembrane proteins (Mizrak et al., 2008). It shows restricted expressionin plasma-membrane protrusions such as epithelial microvilli (Baueret al., 2008). Two antibodies – CD133/1 (also referred to as AC133) andCD133/2 (also referred to as AC141) – recognize different glycosylatedepitopes; most studies use CD133/1. CD133 expression is influenced bycell-cycle status (CD133+ cells are generally in cycle) (Sun et al., 2009;Jaksch et al., 2008; Tirino et al., 2008). In gliomas, the expression ofHIF2 correlates with expression of CD133; thus, hypoxia might blockTPC differentiation (Li et al., 2009).

CD166Activated leukocyte cell-adhesion molecule (ALCAM).

EpCAMA glycosylated type I integral membrane protein expressed by manytumour cells (Munz et al., 2009). Involved in components of Wntsignalling that stimulate cell proliferation after cleavage of EpCAMintracellular domain.

SPSide population; defines cells that are fluorescent-dull by flow-cytometric analysis. SP cells efflux fluorescent dyes, such as Hoechst33342 and DyeCycle Violet, which is a phenotype that usually dependson expression of the ABC superfamily of membrane transporters. The49 human ABC transporters are organized into seven families (A-G;http://www.nutrigene.4t.com/humanabc.htm). In particular, ABCG2(also known as breast cancer resistance protein 1, BCRP1) is expressedin many stem cells and is upregulated in hypoxic environments (such asstem cell niches) (Teramura et al., 2008).

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(phosphatase and tensin homologue; a known tumour suppressorin several contexts) in these cells after androgen-stimulatedregeneration of the epithelium promoted the rapid development ofprostatic intraepithelial neoplasia (PIN) and microinvasion (Wanget al., 2009). Thus, in the prostate, there might be two distinct celltypes from which tumours arise – basal and luminal. In particular,the indication that these cancer-initiating cells are of luminal originis consistent with the absence of basal cells in human prostaticadenocarcinoma.

Mammary and gonadal tissuesThere might also be more than one cell type in breast tissue fromwhich cancer develops. Many breast cancers are believed tooriginate from undifferentiated oestrogen-receptor-negative (ER–)multipotential basal stem cells, the numbers of which can be hugelyexpanded by exposure to progesterone (Joshi et al., 2010), perhapsexplaining why more reproductive cycles (early menarche, latemenopause) are a strong risk factor for breast cancer. However, inbasal-like breast cancers, gene-expression profiling suggests thattumours originate from luminal progenitors. Moreover, luminalprogenitors are expanded in pre-neoplastic tissue from carriers ofmutations in the breast cancer 1 gene (BRCA1) (Lim et al., 2009).Two distinct cell types (one enriched for myoepithelial transcripts)that were differentiated from human breast epithelium andtransfected with the same defined genetic elements produced verydifferent tumour types (Ince et al., 2007). This observation suggeststhat clinical differences between the various subtypes of breastcancer reflect different populations of founder cells. In the testis,pluripotent germ-cell tumours (teratomas) probably arise frommaturation-arrested gonocytes (foetal germ cells), which arephenotypically similar to the probable precursor lesion of teratomas,known as carcinoma in situ (CIS) (Kristensen et al., 2008); theexpression of stem cell factor (SCF) detects CIS cells (Stoop et al.,2008).

LiverTwo distinct cell populations can give rise to hepatocellularcarcinoma (HCC). Because oncogenic transgenes can be drivenby albumin promoters, hepatocytes are implicated in many animalmodels of HCC. However, hepatic progenitor cells (HPCs) thatarise from a potential stem cell compartment in the small biliaryducts proliferate in response to chronic liver damage such ascirrhosis, which usually precedes HCC. Therefore, HPCs mightbe particularly susceptible to mutation (Alison et al., 2009a),and maturation arrest of HPCs might contribute to tumorigenesisin the liver. Four prognostic subtypes of HCC have beenidentified that correspond to a hierarchy of liver-cell lineages(Yamashita et al., 2008). Patients with tumours comprising asizeable proportion of either foetal-like liver cells or HPCs hadthe poorest prognosis, whereas patients with tumours composedof cells resembling either mature hepatocytes or biliary cells hada more favourable clinical outcome. Moreover, gene-expressionprofiling identified a subset of HCCs with a poor prognosis andin which patient tumour-cell profiles suggested an HPC origin(Lee et al., 2006). Finally, identifying tumour cells on the basisof either high or low expression of the HPC marker cytokeratin19 (CK19, also known as KRT19) can be used to identify anHCC patient group with a shorter time to recurrence – that is,high CK19 expression is predictive of local or distant recurrenceafter whole-liver resection and transplantation (Durnez et al.,2006).

LungThe phenotypic diversity of lung tumours, which vary dependingon their location, is probably due to region-specific variation instem cells in the tracheobronchial tree (Alison et al., 2009c). Theidea that pro-oncogenic stem cell niches exist in the lung issupported by mouse models in which tissue-wide knockdown of atumour suppressor gene (e.g. p53) or upregulation of a proto-oncogene under the regulation of a widely expressed lung-specificpromoter (in effect inducing a ‘field cancerization’ effect), doesnot induce a wide distribution of tumours. Instead, tumours aregeographically linked to stem cell niches. For example, mutationsin KRAS are common in human non-small-cell lung cancer(NSCLC) (Riely et al., 2009), but mouse models that havewidespread Kras mutations only exhibit adenocarcinoma in thebronchioalveolar region (Jackson et al., 2001) that involves theexpansion of self-renewing, multipotent bronchioalveolar stemcells at the bronchioalveolar duct junction (Kim et al., 2005).Furthermore, human small-cell lung cancers (SCLCs) arise frommid-level bronchioles where neuroendocrine precursors are located,whereas squamous-cell carcinomas (SCCs) generally occur in theproximal airways and appear to develop from cytokeratin-14-positive basal cells found either in the submucosal gland ducts orintercartilagenous boundaries (Barth et al., 2000).

Haematopoietic cellsThe cell of origin of most haematological malignancies isprobably a multipotential haematopoietic stem cell (HSC). Forexample, in chronic myeloid leukaemia (CML), in which 95% ofaffected patients have the Philadelphia chromosome (which islikely to be the founder mutation), the BCR-ABL oncogenic fusiontranscript can be found in otherwise normal mature blood cellsthat have differentiated from an HSC carrying this translocation(Cobaleda et al., 2000). However, leukaemias might developfrom more committed progenitors that have reacquired the stemcell property of self-renewal (Passegue et al., 2003; Dalerbaet al., 2007a). This idea is supported by the finding thattransfection with oncogenic fusion genes such as MOZ-TIF2 canconfer granulocyte-monocyte progenitors (GMPs) with leukaemicproperties (Chan and Huntley, 2008). Other examples in whichleukaemia is derived from progenitors include CML patientsin blast crisis (the final phase in the evolution of leukaemia), inwhich GMPs can become self-renewing TPCs; this change isassociated with elevated Wnt signalling in the GMPs (Jamiesonet al., 2004). Likewise, in childhood acute lymphoblasticleukaemia, clonal T-cell receptor rearrangements have beenreported to occur in CD133+, CD19+ TPCs, suggesting de-differentiation of a cell that has already gone through the firststages of differentiation (Cox et al., 2009). Recent studies inmice have also highlighted the importance of the niche for stemcell homeostasis, showing that stromal cell dysfunction can leadto leukaemogenesis (Raaijmakers et al., 2010).

Overall, experimental and clinical evidence support the ideathat, in humans, the process of tumorigenesis begins in an adultstem cell, although other more committed cells, particularly in thehaematopoietic system, might also be founder cells of malignancy.Whether the cells that are operationally referred to as TPCs (on thebasis of characteristics determined in an in-vivo assay, see Fig. 2and discussion below) are directly derived from asymmetric orsymmetric division of mutated adult stem cells, or whether theyevolve from further-differentiated cancer cells is presently unclear.In the following sections, we explore the characteristics of TPCs

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and highlight the unique characteristics that differentiate themfrom normal stem cells.

Characteristics of TPCsFunctional assays for TPCsTPCs are believed to self-renew and to give rise to a hierarchy ofprogenitor and differentiated cells, albeit in an unorthodox mannerthat gives rise to more TPCs through symmetric divisions (Bomanet al., 2007; Cicalese et al., 2009) (Fig. 1). Operationally, TPCs area population of cells that can be defined by the expression ofspecific molecules (see Box 1), and that are more tumorigenic thanthe bulk tumour population. An example of a ‘tumorigenicityassay’ for human TPCs that reveals their tumorigenic potential isthe transplantation of human cells into either nude or non-obesediabetic/severe combined immunodeficient (NOD/SCID) mice,which lack major elements of the immune system and therefore donot reject the human cells (Fig. 2C). Such assays can becomplemented by two types of ex vivo assay known asclonogenicity assays (Fig. 2A,B); these assays also seem to beuseful surrogates for in vivo TPC assays.

It was previously thought that TPCs were present in only verysmall numbers, as large numbers of human tumour cells had to bexenotransplanted into immunodeficient mice to generate tumours.However, this might be because the human cells in this assay arein a foreign microenvironment, as transplantation of mouse tumourcells into mice indicates that TPCs can be quite common in sometumours. For example, as few as ten mouse lymphoma or acutemyeloid leukaemia (AML) cells can propagate tumours when theyare transplanted into immunocompetent histocompatible mice(Kelly et al., 2007). Furthermore, using standard immunodeficientNOD/SCID mice, the frequency of TPCs in human melanoma wasestimated to be 1 in 106 (Schatton et al., 2008), but even singlehuman melanoma cells were found to form tumours in more highlyimmunocompromised mice [NOD/SCID mice that were also nullfor the interleukin-2 receptor -chain (IL2R)] (Quintana et al.,2008). Clearly, estimated frequencies of human TPCs in cancercrucially depend on the immune status of the recipient mouse.

Mouse models of cancer support the concept of TPCsSeveral studies have reported subpopulations of TPCs in mousemodels of cancer, overcoming the initial skepticism of experimentsinvolving xenografting human cells into immunodeficient mice. Ina mouse model of HCC (involving targeted deletion of Pten inhepatocytes), only a CD133+CD45– population was found to havetumour-propagating activity in nude mice (Rountree et al., 2009).In mammary tumours developing in p53-null mice, TPCs that wereable to form tumours which expressed both myoepithelial andluminal markers had the phenotype of normal mammary-glandstem cells (CD29highCD24high) (Zhang et al., 2008a), but theirfrequency was only 1:300, supporting the notion that TPCs can bea minority subpopulation. A subset of TPCs has also been identifiedin mouse medulloblastomas occurring in Ptc+/– mutant mice (Readet al., 2009).

EMT can generate TPCsEpithelial-to-mesenchymal transition (EMT) is a process by whichthe largely E-cadherin (CDH1)-dependent cell contacts betweencontiguous epithelial cells break down, resulting in epithelial cellsbecoming fibroblast-like and motile. For example, a main sourceof fibroblasts in renal fibrosis is through EMT of kidney epithelialcells, which is mediated by transforming growth factor 1 (TGF1).

Transcription factors such as Twist and Snail downregulate CDH1expression; accordingly, it has been shown that, when Twist orSnail expression is upregulated in breast cancer epithelial cells,EMT results. Remarkably, the resulting mesenchymal-like cellsacquire a breast TPC phenotype (CD44+CD24low) (Mani et al.,2008). In ovarian cancer, transfection with Snail and Snail2 (alsoknown as Slug) leads to de-repression of ‘stemness’ genes,including Nanog and KLF4, and four- to fivefold increases in thesize of a CD44+CD117+ TPC population that is more resistant tochemo- and radiotherapy (Kurrey et al., 2009).

Undifferentiated tumours that have features of EMT and containlarge numbers of stem cells can also be induced in mouse mammaryglands by overexpressing the developmentally regulated homeoboxprotein SIX1 (McCoy et al., 2009). SIX1 is more commonlyexpressed in human metastatic lesions compared with primarybreast tumours. Another protein associated with aggressive breastcancer is YB-1 (also known as YBX1). YB-1 activates thetranslation of mRNAs that encode proteins such as Snail, Twistand ZEB1, and other transcription factors that coordinate EMT(Evdokimova et al., 2009). Although the rarity of TPCs hashampered efforts to identify drugs that selectively kill them, theacquisition of the breast TPC phenotype by transformed breastcancer epithelial cells (CD44highCD24low) upon EMT has made


Fig. 1. Tumour evolution and heterogeneity. TPCs probably originate fromnormal stem cells (S, yellow), can be derived from transit-amplifying cells(TACs, blue) but not terminally differentiated (TD) cells (green). TPCs canundergo self-renewal by asymmetric cell division and increase in number bysymmetric divisions. TPCs might have a single identity (red outline), butfurther genetic and epigenetic changes can give rise to other TPCs with adifferent phenotype (TPC2, green outline). In addition, EMT might give rise tomore TPCs. This hierarchical organization, which gives rise to intratumoralheterogeneity, might be supplemented by the clonal evolution of a populationof cells with a selective growth advantage (TAC, blue/yellow) (Odoux et al.,2008; Shipitsin et al., 2007).

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high-throughput screening possible (Gupta et al., 2009). In thisstudy, short hairpin RNA (shRNA)-mediated inhibition of CDH1was used to generate large numbers of TPCs, and the antibioticsalinomycin was identified as a highly effective agent to reducetheir viability, clonogenicity and tumorigenicity.

The development of metastasis might involve the disseminationof TPCs, in particular cells at the tumour margins that haveundergone EMT (Brabletz et al., 2009). This process might beaided by the expression of chemokine receptors on TPCs, asobserved in pancreatic cancer (Hermann et al., 2007). Overall,EMT in epithelial tumours appears to be an adverse prognosticfactor and can be associated with acquisition of a TPC phenotype.

Are TPCs multipotential?Are tumours heterogeneous because of distinct clones that arisefrom different TPCs, or are the TPCs in a particular tumourmultipotential – similar to many normal stem cells? Clonalpopulations have been derived from patients with colorectal

cancer (CRC) that recapitulated the heterogeneity of the originaltumours when they were xenografted, exhibiting enterocytic,neuroendocrine and goblet-cell differentiation – all from a singlecell (Kirkland, 1988; Odoux et al., 2008; Vermeulen et al., 2008).In glioblastoma multiforme (GBM), single TPCs can generate allthree lineages (neural, astrocytic and oligodendrocytic; often indifferent proportions), underscoring TPC multipotentiality(Borovski et al., 2009). However, these observations do notpreclude the possibility that there are also different TPCs in atumour. In support of this idea, the centre and peripheral areas ofhuman glioblastomas have been found to contain two populationsof cytogenetically diverse TPCs: both were multipotential, butthey had distinct tumorigenic potentials (Piccirillo et al., 2009b)(although they probably both derived from a common clone).Cytogenetically diverse clones have also been found in humanbreast cancer (Shipitsin et al., 2007) and metastatic colon cancer(Odoux et al., 2008). Each tumour contained consistent (clonal)karyotypic abnormalities, but also contained clones with uniqueadditional abnormalities, suggesting chromosomal instability thatcould contribute to the generation of alternative TPCs – thisclonal evolutionary process gives rise to intratumoralheterogeneity together with that of the TPC hierarchy (Fig. 1).Indeed, chromosomal instability directly generates increasednumbers of tumorigenic side population (SP) cells (see Box 1) innasopharyngeal carcinoma (Liang et al., 2010).

The TPC nicheNormal adult stem cells seem to be regulated by molecular cuesthat are provided by neighbouring connective-tissue cells, mainlymesenchymal (fibroblast-like) cells and vascular cells; these stromalcells contribute to what is known as the stem cell niche (Watt andHogan, 2000; Alison and Islam, 2009). TPCs can be present in andinfluenced by a similar microenvironment. In both SCC (Atsumiet al., 2008) and bladder cancer (He et al., 2009), putative TPCsare located at the tumour-stroma interface. In colorectal cancer, thepromotion of Wnt signalling in TPCs requires co-stimulation byhepatocyte growth factor (HGF) produced by stromal fibroblasts,illustrating the importance of the microenvironment in maintainingthe tumorigenic capacity of cells within tumours (Vermeulen et al.,2010). Brain TPCs identified on the basis of the expression ofnestin and CD133 have been observed congregated close tocapillaries in a niche; thus, therapeutic targeting of the vasculaturecould destroy the niche as well as achieve tumour debulking(Calabrese et al., 2007). In leukaemic mice, leukaemic TPCsoutcompete normal HSCs for their normal osteoblastic and vascularniches and, as a consequence, normal HSCs lack support anddecline in number (Colmone et al., 2008). There is increasingevidence that the leukaemic TPCs have maintenance requirementssimilar to those of HSCs, which are provided by the niche (Laneet al., 2009); therefore therapeutic targeting of the niche occupiedby leukaemic TPCs is an attractive yet difficult proposition. Suchstrategies could include inhibiting leukaemic TPC homing andengraftment by antibody blocking of molecules such as CD44and CXCR4, which are expressed by tumour cells, and CXCL12(also known as SDF-1), which is expressed by niche stromal cells(Fig. 3C).

Markers of TPCs in human tumoursTPCs have been prospectively isolated on the basis of theirexpression of particular markers – often CD133, but also cell-adhesion molecules, cytoprotective enzymes (e.g. aldehyde


Fig. 2. Assays for TPCs. Selected cells (e.g. CD133+ cells) can be assessedeither for their ability to form a large family of descendants, i.e. whether theyare clonogenic in an in-vitro assay for TPCs, or by their ability to initiate newtumour growth after xenotransplantation into immunocompromised mice.(A)Cells can be plated at low density directly onto plastic or in Matrigel and,after several weeks, the plating efficiency (in percent) can be calculated as thenumber of macroscopically visible clones per total number of cells plated; thisassay probably does not distinguish between stem and progenitor cells.(B)Cells can be grown at low density in non-adherent conditions in semi-liquid medium, the resulting spheres (e.g. neurospheres, colonospheres,mammospheres) should generate both CD133+ and CD133– cells. Theresulting CD133+ cells can be serially passaged, generating secondary andtertiary spheres with a cellular composition resembling that of the primaryspheres. (C)The gold-standard assay for TPCs in which the selected cells areorthotopically (common for brain) or ectopically (e.g. subcutaneous site,common for colon) xenotransplanted into an immunocompromised mouse.

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dehydrogenase, ALDH) and drug-efflux pumps (e.g. ABCtransporters) (Box 1). As this field is still evolving, there is notyet an apparent consensus about the best marker by which toidentify TPCs in any particular cancer (Table 1). Brain TPCshave mainly been studied in medulloblastoma, a paediatric tumour

of the cerebellum and gliomas: in this case, CD133 has been thebest marker to enrich for TPCs (Piccirillo et al., 2009a). Bycontrast, there has not been a similar consensus on the bestmarker for identifying TPCs in other cancers. This is particularlytrue for gastrointestinal carcinomas, as several permutations and


Fig. 3. Possible therapeutic targets in TPCs. Bold blocking arrows indicate points of therapeutic intervention. (A)Strategies based on targeting intracellularpathways active in TPCs. The active Wnt, EpCAM, Hedgehog and Delta/Notch pathways have all been implicated in TPC self-renewal and proliferation, and canbe inhibited to therapeutically target these cells. (B)Strategies based on targeting molecules that are highly expressed by TPCs. Targeting ABC transporters andALDH activity might sensitise TPCs to currently available drugs. CD133 is another potential target (although CD133 expression can be ubiquitous in sometissues). Manipulation of mRNA expression levels through microRNAs (miRNAs) is also a promising strategy by which to target TPCs. (C) Strategies based ontargeting molecules involved in invasion and metastasis. Blocking CXCR4-CXCL12 and CD44-hyaluronan (HA) interactions might target TPCs. Inhibiting EMTmight slow the generation of more TPCs with metastatic potential. The direct destruction of endothelial cells that maintain vascular niches also has the potential toeliminate TPCs. -Cat, -catenin; Dsh, dishevelled; ECM, extracellular matrix; EpEX, EpCAM extracellular domain; Fz; Frizzled; GSK3, glycogen synthasekinase 3; HH, Hedgehog; Lrp5/6, low-density lipoprotein receptor-related protein 5 and/or 6; PTC, Patched; SMO, Smoothened.

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apparent contradictions have been proposed regarding the use ofvarious markers, particularly CD133 (Shmelkov et al., 2008).

High ALDH activity has been used to select for TPCs in humanbreast cancer cell lines (Charafe-Jauffret et al., 2009). In primarytumours, when TPCs are selected on the basis of the combined

expression of ALDH and CD44+CD24–/low, as few as 20 of thesecells are usually tumorigenic (Ginestier et al., 2007). Breast cancersthat contain cells with the CD44+CD24– phenotype are mostcommon in basal-like tumours, but not all breast cancers contain asubpopulation of cells that have this phenotype (Honeth et al.,


Table 1. Markers of TPCs in human tumours

Tumour Phenotype Reference

Acute T lymphoblastic leukaemia CD90+CD110+ (Mpl) (Yamazaki et al., 2009)

Acute myeloid leukaemia CD34+CD38- (Bonnet and Dick, 1977) SSClowALDHbright (Cheung et al., 2007)

Breast carcinoma CD44+CD24–ALDH (Ginestier et al., 2007) ALDH (Charafe-Jauffret et al., 2009)

Bladder carcinoma SP (Oates et al., 2009)

Childhood B-acute lymphoblastic leukaemia CD133+CD19–CD38– (Cox et al., 2009)

Colorectal carcinoma CD133+ (Ricci-Vitiani et al., 2007) CD133+ (O’Brien et al., 2007) CD133+ and CD133– (Shmelkov et al., 2008) CD44+ (Du et al., 2008) EpCAM+CD44+CD166+ (Dalerba et al., 2007b) CD44+ALDH1 (Chu et al., 2009) ALDH1 (Huang, E. H. et al., 2009) EpCAM+ALDH1 (Carpentino et al., 2009)

Endometrial carcinoma CD133+ (Rutella et al., 2009) SP (Friel et al., 2008)

Ewing sarcoma CD133+ (Suva et al., 2009)

Gastric carcinoma CD44+ (Takaishi et al., 2009)

Head and neck SCC CD44+ (Prince et al., 2007) ALDH (Clay et al., 2010)

Hepatocellular carcinoma (HCC) CD133+ (Song et al., 2008) CD44+CD90+ (Yang et al., 2008a; Yang et al., 2008b) CD133+ (Ma et al., 2007)

HCC cell lines SP (Chiba et al., 2008)

Hodgkin lymphoma CD27+ALDH (Jones et al., 2009)

Lung carcinoma (NSCLC) CD133+ (Chen et al., 2008) CD133+ (Eramo et al., 2008) SP (Levina et al., 2008) SP (Sung et al., 2008)

Lung carcinoma (SCLC) SP (Das et al., 2008) CD133+ASCL+ALDH1 (Jiang et al., 2009a; Jiang et al., 2009b)

Medulloblastoma, glioma CD133+ (Piccirillo et al., 2009a)

Osteosarcoma CD133+ (Tirino et al., 2008) ALDH (Wang et al., 2010)

Ovarian adenocarcinoma CD44+CD117+ (Zhang, S. et al., 2008) CD44+MyD88+ (Alvero et al., 2009) CD133+ (Baba et al., 2009)

Pancreatic carcinoma CD133+ (Hermann et al., 2007) CD133+ (Maeda et al., 2008) CD44+CD24+EpCAM+ (Li et al., 2007)

Prostate carcinoma CD133+CD44+21+ (Collins et al., 2005) CD44+ (Patrawala et al., 2006) CD44+ (Klarmann et al., 2009) CD44+CD24– (Hurt et al., 2008)

Renal carcinoma CD105+ (Bussolati et al., 2008)

SCC (A431) Podoplanin+ (Atsumi et al., 2008) SP (Loebinger et al., 2008) SP (Zhang et al., 2009)

ASCL1, achaete-scute complex homologue 1; MyD88, myeloid differentiation factor 88; SP, side population; SSC, side scatter.

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2008). Brca1-deficient mouse mammary tumours contain distinctCD44+CD24– and CD133+ tumorigenic subpopulations (Wrightet al., 2008).

The first TPCs were recognized in acute myeloid leukaemia asa small subset of cells that, similar to normal HSCs, had thesignature CD34+CD38– (Bonnet and Dick, 1997). Unlike studiesof solid tumours, among which there is little apparent agreementregarding the identity of TPCs, the consensus view inhaematological malignancies is that the CD34+CD38– signaturedoes identify most TPCs. However, it should be noted that othermarkers have also been used to identify TPCs in these types ofmalignancy (see Table 1).

TPCs in tumour invasion and metastasisIf TPCs are involved in tumorigenesis, it follows that theirfrequency in primary tumours is correlated with the extent oftumour invasion and metastasis and, in turn, to patient prognosis(their clinical outlook). The chemokine receptor CXCR4 is oftenexpressed by tumour cells and by TPCs in particular. Hence, thesecells can migrate along a gradient of the CXCR4 ligand, CXCL12,which can originate from haematopoietic niches and many othertissue sites, thereby facilitating metastasis. For example, CXCR4is highly expressed by human endometrial cancer, whereasCXCL12 is expressed by normal tissues; a CXCR4-neutralizingantibody was effective in blocking the seeding of cancer cells atectopic sites in nude mice, highlighting the potential use ofantagonists of chemokine receptors as a therapeutic strategy(Gelmini et al., 2009) (although it was not clear if TPCs werespecifically targeted in this study). Similarly, blocking CXCR4expression in sphere-forming cells (putative TPCs) in breast cancercell lines using antibodies or shRNA inhibited invasive behaviourin vitro (Krohn et al., 2009). CXCR4-expressing CD133+ TPCs arealso directly involved in the metastasis of human pancreatic cancercells: when CD133+ TPCs were partitioned into CXCR4+ andCXCR4– fractions, only the CXCR4+ cells formed metastases afterorthotopic xenografting. Notably, the metastases could be blockedby AMD3100, a small-molecule inhibitor of CXCR4 (Hermannet al., 2007).

The invasion of ALDH+ TPCs in breast cancer cell lines involvesinterleukin-8 (IL-8) and its receptor CXCR1; these TPCs expressedhigh levels of CXCR1 and showed markedly enhanced invasionthrough Matrigel in response to IL-8 (Charafe-Jauffret et al., 2009).The expression of various integrins and of the transmembraneglycoprotein CD44 on gastric cancer SP cells can also be importantfor metastasis (Nishii et al., 2009).

In the metaplastic type of breast cancer that is characterised byaggressive growth, chemoresistance and poor patient outcome, thegene signature of the tumour generally closely matches that ofCD44+CD24–/low TPCs found in most human breast tumours(Hennessey et al., 2009). As well as illustrating the link betweenTPCs and metastatic tumours, these data also highlight thatstemness traits are usually a poor prognostic indicator.

TPCs and patient prognosisOverall, we believe that, for a given tumour type, a high numberof stem cells indicates a poor prognosis. In breast cancer, forexample, the most poorly differentiated tumours have the highestburden of TPCs (Pece et al., 2010). In brain tumours, high CD133expression is a prognostic marker for reduced time of disease-free survival and overall survival, a measure that is independentof tumour grade, the extent of resection and the age of the patient

(Cheng et al., 2009). Another study reported that combined nestinand CD133 expression is a marker of poor prognosis (Zhanget al., 2008b). Neurosphere formation (an assay to identify TPCs)has also been found to be an independent predictor of death inpatients with malignant gliomas, suggesting that the ability topropagate neurospheres in culture is a clinically relevantparameter (Laks et al., 2009). High CD133 expression incolorectal carcinoma has also been found to be an independentmarker of poor prognosis (Horst et al., 2009). In pancreaticcancer, in which 60% of tumours express CD133 (with <15%positive cells per case), CD133 expression was found to be anindependent adverse prognostic factor for 5-year survival. Inaddition, CD133 expression was significantly associated withlymph-node metastases (Maeda et al., 2008). However, despitethe large body of evidence showing that TPCs in a wide range oftumour types express CD133, the functional role of this moleculein TPC activity is not clear. Although high CD133 expression istightly correlated with colorectal liver metastasis, small interferingRNA (siRNA)-mediated knockdown of CD133 expression incultured colon cancer cell lines affected neither overall cellmigration nor invasion (Horst et al., 2009). High ALDHexpression in tumours has also been associated with poorprognosis in a number of tumour types including breast (Morimotoet al., 2009; Tanei et al., 2009), pancreas (Rasheed et al., 2010)and early-stage lung cancer (Jiang et al., 2009a). High activitylevels of the ABC transporters have also been reported to be asign of poor prognosis; in patients with AML, there is reducedoverall 4-year survival in patients with tumours expressing highlevels of ABCC11 (also known as MRP8) (Guo et al., 2009). Insummary, patients with tumours that express high levels ofmolecules associated with TPCs have a poorer prognosis thanpatients with tumours that express low levels of these markers.

Therapeutic targeting of TPCsIf TPCs are the roots of cancer, then these are the cells that mustbe specifically eliminated for a successful therapy. However, it iswell known that many cancers are resistant to currently availabletherapies. For example, CD133+ TPCs in glioblastomas are highlyresistant to irradiation owing to upregulation of the DNA-damageresponse and can be sensitised by inhibition of Chk1 and Chk2(Bao et al., 2006), whereas the entire pool of CD133+ TPCs can bereduced by forcing them to differentiate into astrocytes with bonemorphogenetic proteins (Piccirillo et al., 2006). Therapy can alsoenrich for TPCs in the residual tumour, as seen with gemcitabinetreatment of pancreatic cancer (Mueller et al., 2009),cyclophosphamide treatment of colorectal cancer (Dylla et al.,2008), doxorubicin and fluorouracil treatment of HCC (Maet al., 2008) and cisplatin, doxorubicin and methotrexate treatmentof lung cancer (Levina et al., 2008). Therefore, highly specifictherapies must be developed to target TPCs.

TPCs and other cancer cells express several potential therapeutictargets (Fig. 3). Targeting key signalling pathways that are activein TPCs is one approach to therapy (Fig. 3A): for example,inhibiting the release of Notch intracellular domain (NICD) with-secretase inhibitors, and by blocking the Hedgehog and Wntpathways with cyclopamine and DKK1, respectively. A newapproach to antagonize Wnt signalling has been to stabilize axin,thereby preserving the -catenin destruction complex (Huang,S. M. et al., 2009). Components of the Wnt signalling pathwayalso link up with the intracellular domain of EpCAM (EpICD) topromote cell proliferation; thus, similar to the Notch pathway,


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targeting the proteases involved in intracellular cleavage arepromising approaches (Munz et al., 2009).

Small-molecule inhibitors of various other molecules expressedby TPCs is an alternative avenue for therapy (Fig. 3B). As the drugresistance of TPCs can often be directly attributed to the activityof ALDH (Dylla et al., 2008) or ABC transporters (Loebingeret al., 2008; Sung et al., 2008), these are both promising targets forsmall-molecule therapy (Fig. 3B). In addition, given that manytypes of cancer cells have a specific microRNA-expression profile(Nicoloso et al., 2009), the use of microRNA-based therapeutictools to target TPCs is an area of increasing interest. For example,low expression of microRNA-199b-5p is a marker of poor patientsurvival in medulloblastoma: this miRNA affects the Notch pathwayby downregulating Hes1 expression, and its introduction reducesproliferation and engraftment of CD133+ medulloblastoma TPCsin athymic mice (Garzia et al., 2009). In GBM, transfection ofmicroRNA-124 and microRNA-137 causes cell-cycle arrest andapparent differentiation of CD133+ cells (Silber et al., 2008).MicroRNA-128 is markedly downregulated in glioblastomas, andintroducing microRNA-128 into glioma cells reduces theirproliferation by directly binding to the 3�-untranslated region ofBMI1 mRNA (Godlewski et al., 2010).

Other therapeutic strategies might involve inhibiting invasionand metastasis by blocking integrins or CD44 (which binds tohyaluronan) at the cell surface (Fig. 3C). For example, CD44+

ovarian tumour cells that express markers of activated pluripotentstem cells might have a selective advantage for spreading throughadhering to the hyaluronic-acid pericellular coat of surroundingmesothelial cells (Bourguignon et al., 2008). Finally, small-molecule inhibitors of CXCR4 were shown to successfully blockthe metastasis of xenografted human pancreatic cancer (Hermannet al., 2007).

As discussed above, EMT of cancer cells can generate more-invasive cells and give rise to new TPCs; therefore, developingtherapies that inhibit this process is an important aim. EMT is aprominent feature of basal-like breast cancer; here, inhibiting Wntsignalling was shown not only to block stem cell self-renewal butalso represses the expression of the CDH1 repressors Twist andSlug, which, in turn, blocks metastasis (DiMeo et al., 2009).

Conclusions and perspectivesNormal adult multipotential stem cells are self-renewing. Ifnormal adult stem cells are the founder cells of many tumours,TPCs probably inherit many of the attributes of normal stemcells. We believe that most human tumours – both solid tumoursand haematological malignancies – have a subpopulation of TPCs,although recent data challenge the once-popular belief that theyare always a minority subpopulation. For example, in malignantmelanoma, they can make up 25% of the total cell population.The fact that mouse tumours also contain subpopulations that areenriched for TPCs supports the notion that malignancies containspecific cells with stem cell attributes. Collective evidenceindicates that markers such as CD133, CD44 and ALDH arecharacteristically expressed by TPCs, and that this populationcan be expanded within tumours by both symmetrical division ofTPCs and/or through other cancer cells undergoing EMT. Manytherapeutic approaches are on the horizon by which to targetTPCs in cancer, which is a challenging prospect given that thesecells seem to be particularly resistant to current therapies.

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Stem cells in cancer: instigators and propagators? · Commentary 2357 Introduction Cancer begins in...

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