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Review article

Cancer stem cells in urologic cancers�

Craig Gedye, Ph.D.c,1, Adee-Jonathan Davidson, M.B.B.S.a,b,1, Martin R. Elmes, M.B.B.S.a,b,Jonathan Cebon, Ph.D., F.R.A.C.P.c, Damien Bolton, M.D., F.R.A.C.S.b,*,

Ian D. Davis, Ph.D., F.R.A.C.P.c

a Ludwig Institute for Cancer Research, Melbourne Centre for Clinical Sciences, Austin Hospital, Heidelberg, Australiab Department of Surgery and Urology, Melbourne University, Austin Health, Studley Road, Heidelberg, Australia

c Department of Medicine, Melbourne University, Austin Health, Studley Road, Heidelberg, Australia

Received 9 April 2009; received in revised form 14 June 2009; accepted 16 June 2009

bstract

There is evolving evidence for the so-called cancer stem cell (CSC) hypothesis, which holds that intra-tumoral heterogeneity in urologicalignancies is hierarchical and that not all cells have the ability to proliferate indefinitely and to generate metastases. This has far-reaching

mplications for research and the clinical management of urologic malignancies. In this review, we outline the tenets and implications ofhe CSC hypothesis, summarize existing evidence for CSCs in urologic malignancies, suggest research directions that may better dissectntra-tumoral heterogeneity in urologic cancers, and outline novel therapeutic modalities implied by the CSC hypothesis. © 2010 Elseviernc. All rights reserved.

Urologic Oncology: Seminars and Original Investigations 28 (2010) 585–590

eywords: Stem cells; Urology

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. Introduction

The management of relapsed and metastatic urologicancers remains suboptimal, and better treatments arelearly required. Existing treatment modalities like cyto-oxic chemotherapy have defined roles such as the measur-ble but modest survival advantage of docetaxel in andro-en-independent prostate cancer, but this treatment is noturative [1]. Even highly active targeted therapies, such asunitinib, now standard of care in the first-line treatment ofetastatic renal carcinoma, do not cure the cancer [2]. A

etter understanding of the biology of urologic malignan-ies may assist in developing novel therapies by definingore relevant treatment targets.

� C.G. is supported by a MOGA/COSA/Roche HOTT Fellowship,ACP CSL Fellowship, and RACP Australia Post Fellowship. A.J.D. is

upported by an Edith-Viola Reid Scholarship. J.C. is a Practitioner Fellowf the National Health and Medical Research Council (NHMRC). I.D.D. isupported by a Victorian Cancer Agency Clinician Researcher Fellowshipnd is an honorary Australian National Health and Medical Researchouncil Practitioner Fellow.

* Corresponding author. Tel.: 61-39457-4049; fax: 61-39457-5829.E-mail address: damienmb@unimelb.edu.au (D. Bolton).

[1 C.G. and A.J.D. contributed equally to this manuscript.

078-1439/$ – see front matter © 2010 Elsevier Inc. All rights reserved.oi:10.1016/j.urolonc.2009.06.010

. The cancer stem cell/tumor-initiating cell hypothesis

Human cancers are composed of a broad variety ofalignant, stromal, immune, and vascular cell types. Evenithin the transformed cell compartment there is consider-

ble morphologic and antigenic variation. Two theoriesccount for this heterogeneity of malignant cells within eachancer. First, the stochastic or clonal evolution model stateshat heterogeneity within cancers is a random process andhat the observed heterogeneity is unrelated to any hierarchymong tumor cells, i.e., every cell within a cancer has amall but non-zero probability of forming a new tumor,epending on niche or external factors (Fig. 1). The alter-ate model is the cancer stem cell (CSC) hypothesis. TheSC hypothesis posits that only a subset of malignantells within each cancer has the ability to self-renew,roliferate extensively, and reproduce the variety of dif-erentiated tumor cells seen within the original cancer.here is now experimental evidence to support the CSCypothesis in many cancers, although it remains contro-ersial [3]. CSCs are functionally defined by their abilityo self-renew, establish tumors, and reproduce intra-tu-oral heterogeneity in serial xenotransplantation assays

4] (Fig. 2).

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586 C. Gedye et al. / Urologic Oncology: Seminars and Original Investigations 28 (2010) 585–590

The CSC hypothesis provides an alternative explanationor treatment failure, with increasing preclinical and clinicalvidence suggesting that resistance to chemotherapy [5] andadiation [6] is intrinsic to the CSC subpopulation. Whilsthese standard treatments may kill the nontumorigenic cellsithin a tumor, CSCs may remain as the “seed” of local

ecurrence or distant metastasis (Fig. 3). CSCs may occupyphysical niche at the leading edge of a growing tumor

butting stroma [7] and vascular [8] structures, consistentith the observation that narrow surgical margins can be

ssociated with an increased risk of local recurrence.CSCs have been demonstrated in an increasing number

f cancers, initially in acute myeloid leukemia [9], and moreecently in solid tumors, such as breast carcinoma [10] andrain neoplasms, [11] amongst many others. Several meth-ds have been employed to identify CSCs. Cell surfacearkers, such as prominin-1/CD133 [12] and the hyaluro-

an receptor/CD44 [13], can identify a subset of cells thatontain CSC in many cancers derived from either epithelialr neuroectodermal tissues. Functional assays can also be

ig. 1. Two theories account for intra-tumoral heterogeneity; the stochasticr clonal evolution model or the hierarchical cancer stem cell (CSC) model,hich postulates that only a subset of cells is able to initiate new tumors.

Color version of figure is available online.)

ig. 2. The “gold standard” experiment to identify tumor-initiating cellsepeatedly form tumors that are identical to the patient’s tumor, whilst non

s available online.)

sed to identify CSC, such as the ability to pump fluorescentyes out of the cell due to the presence of multi-drug effluxumps (the “side population” method [14]) or expression ofhe detoxifying enzyme aldehyde dehydrogenase [15].

Although their signaling pathways and functional prop-rties may be similar to physiological stem cells, and CSCight in some cases be derived from such cells, CSC may

lso arise from progenitor or differentiated cells that reac-uire “stemness” properties. This variability in the “cell-of-rigin” may contribute to inter-patient tumor variability.nfortunately, this cell-of-origin question has led to confu-

ion and disagreement about the CSC hypothesis, and so forlarity the term “tumor-initiating cells” (T-IC), which isqually valid and more accurate, will be employed in thiseview.

Another implication of the T-IC hypothesis is that ther-pies targeting T-IC alone may be most appropriate inatients with minimal residual disease. In contrast, macro-

ig. 3. Treatment-resistant tumor-initiating cells (T-IC) surviving chemo-herapy and radiation may seed local recurrence or distant metastasis (left).f T-IC could be selectively eliminated, nontumorigenic cells may maturend senesce or locally proliferate (right), but could not metastasize. Spe-ifically targeting T-IC will be most useful as an adjunct to conventionalreatments (center). (Color version of figure is available online.)

is the serial xenotransplantation assay. The proposed T-IC (yellow) canancer cells (blue) are unable to form new tumors. (Color version of figure

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copic disease will need to be debulked, as the nontumori-enic cells making up the bulk of the tumor can still causeorbidity. Targeting T-IC alone will not completely replace

xisting treatment modalities effective for nontumorigenicells, and a combination approach may be optimal (Fig. 3).

The T-IC hypothesis suggests novel goals for the treat-ent of cancer, for example by selectively targeting T-IC

ubpopulations, targeting the T-IC niche or encouraging theifferentiation of T-IC to nontumorigenic cells. Few assess-ents of the clinical validity of targeting T-IC have been

ublished to date. Preclinical examples of these novel strat-gies within glioblastoma multiforme xenografts include theirect targeting of L1CAM/CD171 [16], the use of bevaci-umab and erlotinib to target the T-IC vascular niche [8],nd induction of expression of microRNA that cause differ-ntiation of T-IC [17].

. Evidence for tumor-initiating cells in transitionalell carcinoma of the bladder

Early clues to the existence of T-IC in transitional cellarcinoma of the bladder (TCC) can be found in the basalayer of urothelium. Normal bladder urothelium is orga-ized in continuously regenerating layers with normal tissueaintenance following a hierarchical pattern of growth. The

asal layer contains undifferentiated cells, which undergoivision and differentiation, with differentiating cells mov-ng to the luminal surface. Proliferating cells are confined tohe basal and parabasal layers, where the anti-apoptotic genecl-2 is expressed [18]. For this reason, much of the re-earch into TCC T-IC has focused on characterizing cells ofhe regenerative basal layer, examining normal urothelialtem cell markers expressed in this niche such as CD44 andD133 [19].

CD44� cells are found in the basal layer of normalrothelium as well as urothelial carcinoma [20,21]. Theost robust data supporting the existence of T-IC in TCCas recently reported in abstract form [22]. The authors

xamined CD44 expression in a tissue array of over 300ladder TCC by immunohistochemistry, finding that around0% of TCC contained CD44� cells. From freshly exciseduman TCC, a unique tumor-initiating subpopulation ex-ressing CD44 was defined in five independent tumorssing the serial xenotransplantation model. CD44� cellsrom these TCC were 10 to 200 times more likely to formumors compared with CD44neg cells. The authors wentn to examine heterogeneity within different subsets ofD44� TCC, finding a variety of different self-renewalroteins in their active forms, such as nuclear Bmi1, Stat3,r �-catenin. Eighty-five percent of CD44� tumors showedctive Gli1 expression, suggesting that this as a plausibleolecular target. This systematic approach to examining

umor heterogeneity is a benchmark for future studies.Finally, the embryonic stem cell marker OCT3/4 is also

xpressed in human bladder cancer [23]. High levels of m

CT3/4 expression are associated with more rapid tumorrogression and shorter cancer-related survival comparedith moderate/low expression in superficial bladder TCC

24].

. Evidence for tumor-initiating cells in renalell carcinoma

During development, the ureteric bud and metanephro-enic mesenchyme give rise to all cell types in the kidney25], and a single metanephric mesenchymal cell can gen-rate any epithelial cell along the nephron except the col-ecting duct. Early research into RCC tumorigenesis fo-used on the expression of metanephric differentiationntigens, such as vimentin, which are normally only ex-ressed in the metanephric blastema [26]. Re-expression ofhese early embryological differentiation molecules is seenn RCC and in renal biopsies from patients with acquiredystic kidney disease from long term hemodialysis, a groupf patients at higher risk of RCC.

Normal somatic stem cells have also been identified andsolated from adult kidney, with CD133� [27] and CD24�/D133� kidney cells [28] forming tubular structures andxpressing renal epithelial markers after injection into im-unocompromised mice. These CD133� renal progenitor

ells are also thought to contribute to new blood vesselormation in renal carcinoma [29], but these markers haveot yet been prospectively examined for their ability todentify RCC T-IC. Other methods to identify T-IC in RCCnclude the ability to exclude fluorescent dyes (“side popu-ation”) [14], or isolation of cells expressing the cell surfacearker CD105 [30]. The major limitation of these studies is

hat experiments were performed on long-term cloned cellines rather than employing directly xenografted or freshlyxcised human renal tumors.

. Evidence for tumor-initiating cells inrostate carcinoma

The presence of normal adult prostate stem cells is sug-ested by observations that a small number of cells withinhe human prostate possess extensive proliferative capabil-ty and can form glandular structures in reconstituted sys-ems [31,32]. A number of markers have been described todentify these normal prostate stem cells, including cytoker-tin-5 and cytokeratin-18 [33], and stem cell markers suchs �2�1 integrin [34] and CD133 [35]. As in other cancers,hese somatic stem cell markers have in turn been applied inhe search for T-IC in prostate cancer.

The �2�1 integrin is expressed in normal human prostatepithelial basal cells in vivo, and correlates with colony-orming ability and the potential to regenerate a fullyifferentiated prostate epithelium in immunocompromised

ice [34]. CD133� cells within the �2�1

hi population were

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588 C. Gedye et al. / Urologic Oncology: Seminars and Original Investigations 28 (2010) 585–590

hown to have higher proliferative potential and ability toeconstitute a normal prostate gland [35]. The combinationf �2�1 integrin and CD44 expression identifies cells withigh long-term in vitro proliferative potential from prostateancer cell lines grown from freshly excised tissue [36].hese CD44�/�2�1

hi/CD133� cells typically represented.1% of cells in tumors samples. In parallel, other workersemonstrated that “side population” cells and CD44� orD44�/�2�1� cells in prostate cancer cell lines and xeno-rafts have enhanced tumorigenic potential [37–40]. How-ver, these cell surface markers have not been prospectivelyxamined using the gold standard T-IC assay of serial xe-otransplantation described above.

Notwithstanding, bioinformatic analyses of “prostateancer stem cells” have been performed. The gene expres-ion profile of benign prostate epithelial cells and malignantD133� and CD133neg cultured prostate cancer cells wereompared by gene expression profiling [41], demonstrating,or example, that genes involved in development and cellommunication were up-regulated in the CD133� popula-ion, while genes implicated in cell proliferation and cellycle were more highly expressed in the CD133neg popula-ion. A gene expression signature of �2�1

hi/CD133� pros-ate cancer cells compared with �2�1

neg/CD133neg prostateancer cells or prostate cells from benign hypertrophy tis-ues has also been published recently [42]. Five hundredighty-one genes were differentially expressed, and the au-hors highlighted potentially targetable pathways, such ashe JAK-STAT pathway, which was more highly expressedn �2�1

hi/CD133� prostate cancer cells [42]. Glinsky ando-workers have described an 11-gene signature demon-trating that expression of “stemness” markers is associatedith inferior cancer-related outcomes [43], although the

tatistical approach used by that group has been questioned44].

. Evidence for cancer stem cells in germ cell tumors

Germ cell tumors (GCT) of the testis and ovary showigh levels of intratumoral heterogeneity, with mixturesf different histological subtypes the norm. There is notet serial xenotransplantation evidence for an intratu-oral hierarchy in GCT, but this group of malignanciesould seem to be the cancer most likely to originate from

tem cells, as all GCT except spermatocytic seminomasrise from specific lesions in the testis known as intratu-ular germ-cell neoplasia unclassified (IGCNU) or car-inoma in situ [45]. Seminoma, nonseminoma GCT, andysgerminomas have been shown to express pluripotenttem cell markers, such as OCT3/4 [46 – 49], NANOG47,48,50,51], SOX2 [47], UTF-1 and REX-1 [52], andD133 [48]. Gene expression profile studies of IGCNUnd embryonal carcinomas reveal an up-regulation of

reviously silenced transcription factors [53]. Gain of

hromosome arm 12p is consistently seen in GCT [54 –6], and genes on this region encode proteins that regu-ate cell cycle (CCND2) and maintain pluripotency inormal stem cells (NANOG and STELLAR) [57].

. Conclusions and future directions

There is evolving evidence that intratumoral heterogene-ty may be generated by an internal hierarchy peaked byumor-initiating “cancer stem cells,” which in turn impliesovel mechanisms of cancer biology and new potentialreatment targets. However, many questions with regard toumor heterogeneity remain unanswered.

Novel questions must be addressed to substantiate orisprove the T-IC hierarchical model of cancer heterogene-ty in urologic malignancies. Obvious examples are as fol-ows. Is the inevitable progression of metastatic prostateancer to the androgen-independent state due to outgrowthf androgen receptor-negative prostate cancer T-IC? Doeshe transition from superficial TCC of the bladder to inva-ive TCC reflect the acquisition of “stemness” qualities,uch as self-renewal and migration, on a background ofore benign proliferation? Are somatic adult renal stem/

rogenitor cells the likely cell-of-origin for RCC? Can thesebservations be exploited therapeutically? The answers tohese and other questions will become apparent onlyhrough careful exploration of the biology of these cancers,nd through fruitful collaborations between urologic sur-eons, oncologists, and cancer biologists. For example, con-iderable research in prostate carcinoma continues with justhree long-established cell lines: LNCaP, PC3, and DU145.stablishment of novel prostate cancer cell lines is veryhallenging [58]. More routine use of xenograft models andhe establishment of reliable serum-free cell lines will likelyarry the field forward. Systematic tissue acquisition andareful correlation with clinical outcomes data will inevita-ly lead to further advances in the understanding of urologicancers and the definition of new and better treatments.

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