Stem cells in breast tumours- Are they ready for the clinicď€Ą, 2012

8/12/2019 Stem cells in breast tumours- Are they ready for the clinic, 2012

1/13

Review

Stem cells in breast tumours: Are they ready for the clinic?

Matthew P. Ablett, Jagdeep K. Singh, Robert B. Clarke

Breast Biology Group, School of Cancer and Enabling Sciences, University of Manchester, Wilmslow Road, Manchester M20 4BX, UK

Available online 26 April 2012

KEYWORDS

Breast cancerStem cellsTherapy resistanceCancer stem cellsBiomarkers

Abstract The concept of stem-like cells in cancer has been gaining currency over the lastdecade or so since evidence for stem cell activity in human leukaemia and solid tumours,including breast cancer, was first published. The evidence established that sub-populationsof cells identified by antibodies to cell surface markers behaved like developmental stem cellsin their capacity to re-grow the human tumour for several generations in experimentalimmune-deficient hosts. The experiments established that cells with tumourigenic capacityexpressed cancer stem cell (CSC) markers and that activity could also be measured byself-renewal of tumour sphere colonies in culture. In breast and other cancers, there is goodevidence that CSCs are relatively resistant to radio- and chemotherapy indicating that novelCSC-targeted therapies are needed. Several pathways are promising targets in breast CSCs.There are several ways of combating CSC activity including inducing their apoptosis, inhibit-ing stem cell self-renewal to either stop their division or to promote their differentiation, ortargeting the CSC niche that supports them. The first challenge for developing novel CSCtherapies is to ascertain which of these CSC properties is being targeted. The second challengeis to determine suitable CSC biomarkers to measure the efficacy of the novel CSC therapies.We propose using biomarkers as a means to identify and assess CSC activity in clinical trials.This is likely to be demanding but feasible in the near future. Thus, we asked if CSCs are readyfor the clinic, however, the emerging question becomes: is the clinic ready for cancer stemcells?2012 Elsevier Ltd. All rights reserved.

1. Introduction

Over the last 8 years, there has been increasing evi-dence for the existence of a cellular hierarchy in manytumour types. These data have emerged from the inves-tigation of a developmental biological paradigm of

tumour behaviour, which proposes that tumour cells,like normal tissue cells, are organised such that stemcells present in low numbers produce numerous differen-tiated cells which make up the bulk of the cancer. Nor-mal breast epithelial tissue consists mainly of eitherluminal cells destined to produce milk or basal myoepi-thelial cells that have a contractile function during lacta-tion. In tumours, differentiation is patently aberrantcompared to the normal tissue with very few humantumours containing myoepithelial differentiation and

0959-8049/$ - see front matter 2012 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.ejca.2012.03.019

Corresponding author:Tel.: +44 161 446 3210; fax: +44 161 4463109.

E-mail address:[email protected](R.B. Clarke).

European Journal of Cancer (2012) 48, 21042116

A v a i l a b l e a t w w w . s c i e n ce d i r e c t . c o m

j o u r n a l h o m e p a g e : w w w . e j c o n l i n e . c o m
http://dx.doi.org/10.1016/j.ejca.2012.03.019mailto:[email protected]://dx.doi.org/10.1016/j.ejca.2012.03.019http://dx.doi.org/10.1016/j.ejca.2012.03.019http://dx.doi.org/10.1016/j.ejca.2012.03.019http://dx.doi.org/10.1016/j.ejca.2012.03.019http://dx.doi.org/10.1016/j.ejca.2012.03.019http://www.sciencedirect.com/http://www.sciencedirect.com/http://dx.doi.org/10.1016/j.ejca.2012.03.019http://dx.doi.org/10.1016/j.ejca.2012.03.019http://dx.doi.org/10.1016/j.ejca.2012.03.019mailto:[email protected]://dx.doi.org/10.1016/j.ejca.2012.03.019


2/13

only partial luminal differentiation (no milk expressed).On the other hand, an infrequent sub-population ofhuman breast tumour cells expressing surface markerssimilar to normal breast epithelial stem cells can be iden-tified and enriched by flow cytometry. Importantly, theirtransplantation into immune-compromised mouse mod-els has demonstrated that they are capable of growinghuman breast tumours. These findings have severalimplications for the understanding of human breast can-cer biology and a major impact on treatment paradigmsin both curative and palliative settings.

2. Evidence for the identification of stem-like cells in

human breast cancers

The cancer stem cell hypothesis posits that cancersare maintained and re-populated by stem-like cellswithin the tumour, termed cancer stem cells (CSCs). Awidely accepted definition of a CSC is . . .a cell within

a tumour that possesses the capacity to self-renew andto cause the heterogeneous lineages of cancer cells thatcomprise the tumour.1

The first strongin vivoevidence in support of the CSCconcept came from classical implantation studies inhuman leukaemia by Bonnet and Dick.2 They used fluo-rescence-activated cell sorting (FACS) to isolate a spe-cific population of cells from acute myeloid leukaemia(AML) patients which were able to initiate AML follow-ing implantation into non-obese diabetic mice withsevere combined immunodeficiency (NOD/SCID). Theleukaemia-initiating cells were defined by expression of

the cell surface antigen CD34 and displayed self-renewal, differentiative and proliferative capacities simi-lar to normal haematopoietic stem cells.

The first evidence for the existence of CSCs in solidhuman tumours came from studies in breast cancer.3

Al-Hajj and colleagues prospectively isolated a tumouri-genic population of cells from primary human breastcancers using FACS based on the phenotype ESA+/CD44+/CD24/low/lineage-. When injected into themammary fat pads of NOD/SCID mice, as few as 200cells with this phenotype consistently formed tumourswhereas 20,000 CD44+/CD24+ cells failed to formtumours and 10,000 unsorted cells only formed tumours

in 25% of cases. Enhanced tumour forming capacity ofESA+/CD44+/CD24/low/lineage- cells has subse-quently been reported by many other groups,4,5 howeverpurification of a single breast CSC able to initiate atumour is lacking. Some tumour biologists argue thatthe CSC hypothesis is too simplistic and propose a morecomplex model of cancer development, termed clonalevolution.6

The clonal evolution model proposes that tumoursarise from an aberrant normal cell clone which prolifer-ates uncontrollably due to accumulation of geneticmutations. The progeny of this clone would accumulate

further mutations,hence the heterogeneous cellular nat-ure of tumours.7,8 Critics of the CSC hypothesis claimthat the current gold standard method for assessingCSCs by injecting cells into immuno-compromised micemay introduce a selection bias for human cells capableof surviving and proliferating in the mouse microenvi-ronment with foreign growth factors and cytokines.9,10

Recently it has been proposed that a merging of the clo-nal evolution model and the CSC hypothesis mayexplain more fully how cancer is maintained and pro-gresses. This merged model predicts that the frequencyof CSCs in each patient will vary dramatically and bedependent on the type of breast cancer and the domi-nant mutations, gene amplifications and deletions. Fur-thermore, dominant clonal CSCs could emerge duringtumour progression as resistant CSCs are preferentiallyselected for by current therapy. It is therefore importantto develop reliable methods to identify and isolate breastCSCs in the clinical setting.

3. How do we identify breast cancer stem cells and

measure their activity?

The current gold standard method of assessingbreast CSC activity is the ability of these cells to re-growtumours following injection into immuno-compromisedmice. A successful enrichment of a CSC population isdemonstrated by significant improvement in tumourige-nicity compared to unsorted cells of the original popula-tion. One could argue that this approach assays for stemcell activity or a state of stemness since a CSC entity

has not been successfully isolated at the single cell level.Two methods currently used to enrich for breast CSCactivity include FACS using antibodies to specific cellsurface markers or intracellular enzymes such as alde-hyde dehydrogenase (ALDH), using the ALDEFLUORassay, and the mammosphere assay. The mammosphereassay is a non-adherent in vitro colony forming assayoriginally developed by Dontu et al.11 Under thesenon-adherent culture conditions differentiated cellsundergo cell death (anoikis) whereas cells with self-renewal capacity are able to survive and proliferateforming individual colonies termed mammospheres.Both the mammosphere assay and tumour forming

capacity in vivo may measure stem cell activity; by defi-nition, they do not measure dormant stem cells. This isan important distinction that researchers need toacknowledge when studying CSCs, because lack ofdetectable CSC activity does not necessarily equate toeradication of the CSC population. This has importantclinical implications because dormant CSCs couldpotentially become activated at a later date thereby lead-ing to disease recurrence.

To date, there is no universal cell surface antigen, orcombination of antigens, for the purification of breastCSCs by antibody techniques. This may be because of

M.P. Ablett et al. / European Journal of Cancer 48 (2012) 21042116 2105


3/13


4/13

chemotherapy was 14-fold greater compared to cellsderived from chemotherapy naive patients.21 Further-more mammospheres from chemotherapy treated

patients could be passaged up to 10 times, whereas theself-renewal capacity of mammospheres from chemo-therapy-naive patients became exhausted after threepassages. Importantly, by studying paired breast speci-mens of seven patients before and after neo-adjuvantchemotherapy the investigators demonstrated a signifi-cantly greater mammosphere forming ability and a9.5-fold increase in the proportion of CD44+/CD24/low

cells after chemotherapy.21

A clinical study involving 31 patients by Li et al.(2008) also demonstrated enrichment of putative breastCSCs following a 12 week course of neoadjuvant che-motherapy with docetaxel or doxorubicin combined

with cyclophosphamide22 They showed that chemother-apy increased the proportion of CD44+/CD24 cells by3-fold and significantly increased the number of cellsable to generate mammospheres 4-fold. By establishinghuman breast cancer xenografts from patients beforeand after treatment they showed that chemotherapydoubled tumour forming capacity in SCID/Beige micesuggesting an enrichment of CSCs. Using a gene expres-sion signature common to both CD44+/CD24/low andmammosphere forming cells, Creighton et al. (2009)showed that these signatures were more pronounced intumour tissue from 12 patients following neoadjuvant

treatment with docetaxel.23 Together these studies sug-gest that breast CSCs are relatively chemo-resistant,and such therapy can enrich for this cell population.

Potential chemo-resistance mechanisms in CSCs includeincreased expression of antiapoptotic proteins, increaseddrug efflux transporters and increased efficiency of DNArepair.24 Furthermore, it is possible that CSCs are lesssensitive to antimitotic agents due to a low rate of pro-liferation. Chemotherapy may thus destroy non-CSCs ina proportion of tumours, leaving the CSCs to poten-tially re-seed disease.

4.2. Radio-resistance

Philips et al. (2006) demonstrated that mammo-spheres derived from MCF-7 and MDA-MB-231 cell

lines were more radio-resistant than cells grown asmonolayer cultures.13 More importantly they demon-strated that fractionated doses of irradiation increasedthe proportion of tumour-initiating CD44+/CD24/low

cells in the non-adherent cell populations of MCF-7monolayer cultures. The authors proposed that reducedinduction of reactive oxygen species (ROS), reduceddouble strand DNA breaks and induction of theNotch-1 pathway were responsible for the observed rel-ative radio-resistance of breast CSCs and greatertumour re-growth during radiotherapy treatment gaps.Similarly Diehn et al. (2009) proposed that CSCs

Table 2Summary of preclinical, animal model and clinical evidence of breast cancer stem cell (CSC) resistance to chemotherapy, radiotherapy andendocrine therapy.

CSC isolation technique Source Type of treatment Enrichment or preferentialsurvival

Reference

Chemo-resistance

ESA+CD44+/CD24low Cell lines 5-Flurouracil or Paclitaxel In vitro FACS 530-fold " 5CD44+/CD24- Cell lines Doxorubicin-selected MCF7s In vitro FACS 30% " and "

tumours in vivo

103

CD44+

/CD24

mammosphereassay

Clinicalsamples

Neoadjuvant 5-flurouracil,epirubicin & cyclophosphamide

In vivo FACS 9.5-fold "In vivoMS " 0.5% to 5.9%

21

CD44+/CD24 mammosphereassay

Clinicalsamples

Neoadjuvant docetaxel ordoxorubicin & cyclophosphamide

In vivo FACS " 5% to 14%

In vitroMS " 5-fold22

CD44+/CD24/mammospheregene expression signature

Clinicalsamples

Neoadjuvant docetaxel In vivo" gene signaturefollowing chemotherapy

23

Radio-resistance

Mammosphere assay Cell lines Radiation single & fractionated In vitro clonogenic assay 2-fold " in survival

13

CD44+/CD24/low Cell lines Radiation fractionated In vitro FACS, up to 3-fold"

104

Sca1+ BALB/c mice Mouse Radiation single dose In vivo FACS 3-fold " 26CD24+Thy1+Lin MMTV-Wnt1

Mouse Radiation fractionated In vivo FACS, 2-fold " 25

LinCD29+CD24+ P53-nullmouse

Mouse Radiation single dose In vitro clonogenic assay upto 10-fold "

105

Endocrineresistance

CK5+ Cell lines Tamoxifen or Fulvestrant In vitro- up to 3.4-fold " inCK5 protein expression

32

Clinicalsamples

Neoadjuvant tamoxifen +/exemestane

In vivo 2-fold " in CK5expressionIn vivo number ofCK5 + cells/field " 2.6 to 30.4

CD44+/CD24/Mammospheregene expression signature

Clinicalsamples

Neoadjuvant letrozol In vivo" gene expressionsignature

23



5/13

contain lower ROS due to increased expression of freeradical scavenging systems.25 Depletion of ROS scav-engers using pharmacological inhibitors decreased clo-nogenicity and resulted in radiosensitisation.25

Using the Hoechst dye efflux method, Woodwardet al. (2007) found that the side population in hyperplas-tic tissue from mouse mammary tumour virus (MMTV)driven Wnt-1 transgenic mice was more than 2-foldgreater than matched controls and could also be selec-tively enriched following exposure to radiation.26

Together these studies provide evidence that breastCSCs are endowed with mammary stem/progenitor cellproperties which confer protection from radiation-induced cell death. This radiation resistant populationis thereby liable to re-seed tumours during and afterfractionated radiotherapy.

However a recent study suggests that the problem ofradio-resistant CSCs may be overcome by localisedhyperthermia therapy using nanoparticle technology.27

Using genetic mouse model of breast cancer and human

breast cancer xenograft mouse models, Atkinson et al.(2010) showed that radio-resistant CSCs could beradio-sensitised by hyperthermia therapy resulting in agreater reduction in tumour size and reduction of self-renewal, measured by serial transplantation studies.27

Although these findings are promising, the clinical effi-cacy of combining radiotherapy with nanoparticlehyperthermia therapy is yet to be determined.

4.3. Endocrine-resistance

Oestrogen deprivation is an important therapeutic

strategy in the management of breast cancers thatexpress the oestrogen receptor-a (ER-a). However,despite an initial response to endocrine therapy, 25%of patients with early breast cancer and all patients withmetastatic breast cancer eventually become resistant totherapy.19 In the normal mouse mammary gland thestem/progenitor cell population is reported to be ERnegative (ER).28 Using CD24lo and CD29+ to sortfor stem cells Asselin-Labat et al. (2006) demonstratedthat less than 0.01% of these cells expressed ER com-pared to 37% of luminal cells.28 Sleeman et al. (2007)subsequently showed that the greatest in vivoclonogeniccapacity lies within the ER/CD24lo cell population.29

In contrast to the normal mouse and human breastwhere there is a dissociation of ER expression and prolif-eration, actively dividing ER + cells are frequentlyobserved in breast hyperplasia and breast cancer.30 Thelevel of ER expression is predictive of response rate toendocrine therapy. However, the high rate of relapseand poor response in ER + metastatic breast cancerpatients suggests that endocrine therapy does not targetthe cells driving tumourigenesis. For example, using theER+/PR + human breast cancer cell line T47D, Horwitzet al. (2008) showed that within the tumour-initiating cell

fraction resides a rare population of ERPRCK5 + cells.31 In vitro, oestrogen combined with tamox-ifen or fulvestrant led to a decrease in ER expression, butan increase in CK5 expression compared with oestrogenalone, suggesting that anti-oestrogen therapy enriches forthe ER-/CK5 + phenotype.32 In a clinical study usingpaired tissue samples, Kabos et al. (2008) reported thatneoadjuvant endocrine therapy resulted in a decrease inER gene expression and an increase in both CK5 geneexpression and the number of CK5 + cells in post-treat-ment tumours compared to pre-treatment tumours.32

Thus, an emerging mechanism of resistance to ER-targeted therapy is the presence of an ER- CSC popula-tion which can differentiate to form treatment-sensitiveER + cells, but which can escape endocrine treatmentand survive to subsequently repopulate the tumour.

Another proposed mechanism of endocrine resistance isvia enhanced growth factor tyrosine kinase signalling path-ways. An inverse relationship between ER and epidermalgrowth factor receptor (EGFR)/HER2 expression has been

demonstrated in cell line models of acquired endocrineresistance and patients. Using an endocrine resistantMCF-7 cell line McClelland et al. (2001) demonstrated thatas endocrine resistance develops, EGFR expressionincreases in parallel with a decrease in ER expression.33

Similarly, using tamoxifen-resistant MCF-7 cell lines,Knowlden et al. (2003) demonstrated an increase in expres-sion of HER2 compared to wild type MCF-7 cells.34 Inboth studies cell proliferation was reduced by targetingthe EGFR/HER2 signalling pathways. Clinical studies inpatients with recurrent breast cancer have demonstratedthat expression of EGFR/HER2 can be associated with

decreased sensitivity to endocrine therapy and a poorerprognosis.35

The acquisition of enhanced EGFR/HER2 signallingand endocrine resistance could potentially arise from theselectionof more stem-like cells.36,37 Using the ALDEFLU-OR assay, Korkaya et al. (2008) demonstrated that over-expression of HER2expanded the proportion of cells withstem-like properties.38 Furthermore, using HER2 over-expressing breast cancer cell lines, Magnifico et al. (2009)showed that mammosphere-forming breast cancer cellshad higher expression of HER2 compared with cells cul-tured under differentiating conditions.39

HER2 over-expression is associated with increased

expression of genes known to be important in regulatingstem cell function and is under the control of Notch1.38,39 Over-expression of HER2 in breast CSCsincreased their invasive capacity and tumourgenicitywhen transplanted into NOD/SCID mice.38 Moreover,inhibition of HER2 significantly decreased the propor-tion of breast CSCs, decreased tumour forming capacityand decreased invasion.38 Together these studies indi-cate that breast CSC activity is regulated by HER2and that this population can be depleted by HER2 inhi-bition. A pre-surgical window trial by Li et al. (2008)

2108 M.P. Ablett et al. / European Journal of Cancer 48 (2012) 21042116


6/13

demonstrated that treatment with lapatinib (a dualEGFR/HER2 inhibitor) led to a decrease in theCD44+/CD24lo CSC fraction and mammosphere form-ing efficiency of the residual tumour.22 Targeting theCSC population in HER2 overexpressing breast cancersmay partially explain the therapeutic success of anti-HER2 therapy using trastuzumab (Herceptin). Howeverthere is also evidence that HER2inhibition may be ben-eficial in HER2 negative cancers.40 A synergistic effect ofchemotherapy and trastuzumab is supported by clinicalstudies which have shown a significant improvement in3 year disease-free survival of HER2 positive patientstreated with trastuzumab combined with conventionalchemotherapy.41,42

5. How do we target breast CSCs?

As described above, there is good evidence that moststandard therapies target the bulk of tumour cells reduc-

ing tumour size, but often do not eliminate CSCs whicheventually may lead to tumour recurrence. It is vital todevelop agents which target the CSCs specifically. Stan-dard therapies in combination with CSC-targeted thera-pies may potentially provide a more effective treatmentstrategy by de-bulking the tumour mass and preventingrecurrence. Combination therapy would also overcomethe possibility that tumour cells could de-differentiateto a more stem-like phenotype induced by extrinsic fac-tors, as recently demonstrated in cell lines.43,44 There areat least three potential ways to target breast CSCs: (1)inhibition of self-renewal signalling pathways therebyinducing differentiation or apoptosis, (2) targeting resis-

tance mechanisms or (3) targeting of the CSC niche(Fig. 1bd).

Monoclonal antibodies raised against specific compo-nents of signalling pathways or cell surface antigenspresent on CSCs have been used to target these cells spe-cifically (Fig. 1b). For example, novel therapeutic anti-bodies targeting the Notch signalling pathway andstem cell surface antigens EpCAM and CD44 are cur-rently in clinical trials (Table 3).

5.1. CSC signalling pathways

Targeting specific components of the Notch signal-ling pathway has proven to be an effective treatmentagainst breast CSCs in pre-clinical studies both in vitroandin vivo. The Notch pathway is implicated in mainte-nance of both normal and malignant stem cell activ-ity.45,46 Aberrant activation of this pathway has beenreported in breast cancer, specifically in the breastCSC population.36 Studies in pre-invasive ductal carci-noma in situ and invasive breast cancer cell lines andclinical samples have shown that inhibition of the Notchsignalling pathway using a c-secretase inhibitor, DAPT,significantly reduces CSC activity.18,36 DAPT prevents

the cleavage and nuclear translocation of the intracellu-lar domain of the Notch receptor, thus abrogatingNotch signalling. The c-secretase inhibitors MK-0752(Merck) and RO4929097 (Roche) are currently in phaseI/II clinical trials in breast cancer47 (Table 3).

Aberrant Hedgehog signalling has been demon-strated in many different types of cancer and is anotherpotential CSC therapeutic target. Hedgehog signallingregulates stem cell fate and proliferation duringdevelop-ment as well as in adult mammalian systems.48 In breastcell lines, Liu et al. (2006) showed that members of theHedgehog signalling pathway (PTCH1, Gli1 and Gli2)were highly expressed in normal and malignant stemcells.49 Expression was down-regulated when the cellswere induced to differentiate. A current clinical studycombines the Roche Notch inhibitor RO4929097, witha Hedgehog inhibitor from Genetech, GDC-0049, basedon evidence that both of these pathways play an impor-tant role in self-renewal and also interact with eachother.47,50

Some unorthodox treatments for breast cancer haveshown the importance of other signalling pathways inbreast CSC activity. The dietary polyphenols, curcuminand piperine have been shown to modulate self-renewalof breast CSCsin vitrousing the mammosphere assay.51

Both of these compounds inhibited Wnt signalling,which has been shown to be dysregulated in many can-cers including breast cancer. Previous studies demon-strated the importance of the NF-kappaB pathway inleukaemia stem cell survival.52,53 This work has beenextended to breast CSCs by Zhou et al. (2008), whofound that three different inhibitors of the NF-kappaB

pathway preferentially inhibited mammosphere forma-tion and proliferation of theside population of MCF-7 cells sorted using FACS.37

Epidemiological studies indicate that diabetes is asso-ciated with an increased risk of breast cancer.54 Metfor-min is commonly used to treat type2 diabetes and hasbeen shown to reduce cancer risk.55 In vivo, metforminreduces tumour growth of triple-negative breast cancercell lines.56 When combined with conventional therapysuch as doxorubicin or tratuzumab, metformin appearsto further suppress tumour growth by selectively target-ing the breast CSCs.5759

5.2. Differentiation therapy

Another method to target CSCs is to induce them todifferentiate, and thus lose their self-renewal potential.To achieve this, the CSCs must exit their quiescent stateand become actively cycling in order to divide and differ-entiate. Differentiation therapy has been used to treatsome haematological cancers successfully. Rexinoidsand retinoids, strong inducers of differentiation, arethe current standard treatment for some types of leukae-mia and have been shown to promote differentiation of



7/13

Traditional therapyTraditional therapy(a)

Loss of CSC re-growsRelapsetumour bulk tumour

CSC therapies alongside traditional therapyp g py

Loss of CSC and

diff ti t d Tumour shrinkage with

(b)

differentiated

cancer cells

Tumour shrinkage with

traditional therapy

Cure

Targeting CSC resistanceTargeting CSC resistance

(c)

Differentiation Tumour shrinkage with

traditional therapy

Cureof CSC traditional therapy

Cure

Targeting CSC self-renewal

L f CSC d(d)

L f CSC i h

Loss of CSC and

tumour shrinkage with

CureLoss of CSC niche

g

traditional therapy

Cure

Targeting CSC niche Cancer Stem Cell Differentiated Tumour Cell Endothelium Fibroblast Collagen Fibresg g g

Fig. 1. The possible effects of different cancer stem cell (CSC) therapies on tumour growth. Traditional therapy (a) targets the differentiated cellsbut not does affect all of the CSCs, leading to tumour relapse. Three strategies for CSC therapy are shown: (b) targeting CSC resistance, (c)targeting CSC self-renewal pathways or (d) targeting components of the CSC niche. Each of these strategies would be predicted to reduce thecapacity for the CSCs to self-renew and repopulate the tumour. Use of these CSC therapies alongside traditional therapies should further reducebreast cancer recurrence rates.

Table 3Clinical progress of novel agents currently in development for the treatment of breast cancer. These agents target specific components of pathwaysor surface integrins shown to be important in breast cancer progression. All agents listed and their clinical status refers to their use in trialsconcerning breast cancer only.

Target Agent Company Clinical status Reference

Notch MK-0752 c-secretase inhibitor Merk Phase I/II 47RO4929097 c-secretase inhibitor Roche Phase I 47Dll4 specific Abs N/A Pre-clinical 106

Hedgehog Cyclopamine SMO antagonist N/A Pre-clinical 107GDC-0449a Genetech Phase I 47

Bcl-2 G3139 antisense oligodeoxynucleotide Genasense Phase I/II 79CD44 P245 CD44 specific Ab N/A Pre-clinical 81EpCAM MT110 EpCAM specific Ab Micromet Phase I www.micromet.de

MT201 (Adecatumumab) Micromet Phase II www.micromet.deEpCAM specific Ab

RAR & RXR Alitretinoinb pan-RAR, pan-RXR N/A Phase I 63ATRAb N/A Phase I/II 61

PARP Olaparib (AZD-2281) PARP inhibitor Astra-Zenecaase Phase I 72Veliparib (ABT-888) PARP inhibitor Abbott Phase II 73

Dll4 delta like 4; Ab antibody; SMO smoothened homologue; EpCAM Epithelial cell specific adhesion molecule; RAR retinoic acidreceptor; RXR rexinoid receptor; ATRA all-trans retinoic acid; PARP Poly (ADP-ribose) polymerase; N/A not applicable, as these agentsare still at the pre-clinical stage or commercially available.a Used in combination with Roche Notch c-secretase inhibitor RO4929097.b Used in combination with tamoxifen.

http://www.micromet.de/http://www.micromet.de/http://www.micromet.de/http://www.micromet.de/


8/13

breast CSCs in vitro.60 These agents have also shownsome promising results in clinicaltrials for the treatmentof breast cancer (Table 3).6163 Breast CSCs displayincreased ability to efflux drugs due to high levels ofATP-binding cassette (ABC)transporters and multidrugresistance (MDR) proteins.64 Side population studieshave shown that breast cancer cells capable of effluxingthe fluorescent dye Hoechst are enriched for CSCs.65,66

Schinel et al. (1994) demonstrated that transgenic micelacking specific ABC transporters showed increaseddrug sensitivity.67 Thus, inhibition of drug transportersmay provide a novel therapeutic means of re-sensitisingCSCs. However, drug transporters such as BCRP andABCG2 (both ABC transporters) have been identifiedon the blood brain barrier thereby conferring a risk ofadverse neurotoxic effects if administered with currentcancer therapies.68,69

5.3. Targeting DNA repair mechanisms and apoptotic

resistance

The ability of a stem cell to maintain genomic andchromosomal stability over time requires highly activeDNA repair mechanisms. We have listed many examplesof CSC resistance to both chemotherapy and radiother-apy (Table 2). This intrinsic resistance can be attributedto enhanced DNA repair mechanisms, upregulated cellcycle control and increased free-radical scavengers.70,71

Compounds aimed at disrupting repair mechanismsmay re-sensitise CSCs to chemotherapy or radiotherapy.In breast cancer, clinical trials are currently underway

using inhibitors of an enzyme responsible for DNArepair, Poly (adenosine disphosphate-ribose) polymerase(PARP). Phase II trials have demonstrated significantimprovement in response rate, tumour shrinkage andoverall survival when PARP inhibitors are used to treattriple negative, metastatic breast cancer, either alone, orin combination with conventional chemotherapy72,73

(Table 3). However, the first Phase III clinical trial tocombine the PARP1 inhibitor, Iniparib (BSI 201), withchemotherapy showed no significant improvement com-pared to chemotherapy alone.74 It has been suggestedthat the poor efficacy of Iniparib in this trial was due toinclusion of PARP-negative breast cancers in the cohort

and future trials should take PARP expression intoaccount.75

As well as targeting DNA repair mechanisms, CSCscould be sensitised by inhibiting pathways involved inapoptotic resistance, such as NF-kappaB and Bcl2. Tar-geting these anti-apoptoticproteins may improve treat-ment of ER cancers.76 Breast CSCs, identified byCD44+ expression, have higher expression of Bcl-2 thandifferentiated cells in human primary breast cancer sam-ples.77 The tolerability of chemotherapy in combinationwith a Bcl-2 antisense oligodeoxynucleotide oblimersen(Genasense, G3139) in neo-adjuvant systemic treatment

of primary breast cancer has been shown in phase Iclinical trials.78,79 This gene-silencing strategy to targetBcl-2 shows promise in the development of apoptosis-modulating therapies.

5.4. CSC microenvironment

The significance of the CSC microenvironmentshould not be overlooked in the pursuit of CSC-targetedtherapies. However, there are few therapies currently indevelopment to specifically target the CSC niche(Fig. 1d). The notion of targeting components of theCSC niche is not new. Miyake et al. (1990) demon-strated that anti-CD44 inhibited the growth of murinebone marrow progenitorsin vitroby preventing interac-tion with hyaluronic acid in the extra cellular matrix.80

Since then, interest in CD44 as a CSC target in manydifferent types of cancers, including breast cancer, hasgrown. Marangoni et al. (2009) examined the effects of

a monoclonal antibody against CD44 (P245) on humanbreast cancer xenografts, with and without conventionalchemotherapy.81 P245 dramatically inhibited the growthof human breast cancer xenografts when given alone,however the tumours re-grew when treatment wasstopped suggesting a cytostatic effect of anti-CD44 treat-ment without complete eradication of all malignantcells.81 However when given in combination with adria-mycin and cyclophosphamide tumour re-growth wasdecreased demonstrating the therapeutic benefit of tar-geting cells which make up the bulk of the tumour incombination with CSC-targeted treatment.81

Integrins also mediate cell-extracellular matrixinteractions and are key cell surface proteins used forenriching breast CSCs.82 Elevated levels of the intracel-lular signalling mediator, focal adhesion kinase (FAK),have been linked with increased invasion.83 Upregula-tion of FAK in ductal carcinoma in situ(DCIS) suggeststhat it is not limited to the invasive phenotype.84 Adirect link between integrin signalling through FAKand breast CSCs has been suggested by Luo et al.(2009). They demonstrated that ablation of FAKreduced the number of CSCs in primary tumoursobtained from FAK-knockout mice.85 A link betweenFAK signalling and breast CSC maintenance, tumour

progression and metastasis has been shown by severalother groups.8688 Although there are several FAKinhibitors in clinical trials, none of these are currentlyin use to treat breast cancer.89

These data suggest that targeting CD44 or integrinrelated proteins associated with the CSC niche (e.g.FAK) may provide an alternative strategy in breast can-cer therapy. Many of the self-renewal pathways men-tioned above play a role in the CSC niche. The Notch,Hedgehog and Wnt signalling pathways all requirecell-to-cell contact for canonical signalling to occur.Therefore these pathways should also be investigated



9/13

when considering novel therapies aimed at targetingcells within the CSC niche.

In addition to intrinsic factors, extrinsic factors withinthe tumour microenvironment can also regulate breastCSC activity and thus may represent novel therapeutictargets. Recent evidence suggests that cytokines such asstromal derived factor-1, interleukin-6 (IL-6) andinterleukin-8 (IL-8) are important in regulating CSCactivity and may play a role in treatment resistance asrecently demonstrated by Max Wichas group in the caseof IL-6 and trastuzumab resistance.9093 Studies usinghuman breast cancer xenograft mouse models haveshown that inhibition of IL-8 signalling specificallyreduces CSC self-renewal, tumour growth and metasta-ses.94 In addition to autocrine production of cytokineswhich promote CSC activity, other cells within thetumour microenvironment, such as fibroblasts, macro-phages and mesenchymal stem cells can also secrete thesecytokines which may further promote CSC activity viaparacrine signalling.95 Clinical evaluation of cytokine

receptor inhibitors is needed to determine the efficacyof targeting these signalling pathways.

6. How can we measure breast CSCs in clinical trials?

Breast cancer clinical trials can be divided into neoad-juvant therapy trials, pre-surgical window trials andadjuvant therapy trials. In order to assess breast CSCsin clinical trials, we need to develop novel therapeuticendpoints which reflect their expression and/or activ-ity/function (see Figs. 2 and 3). Fig. 2 highlights the

need for reassessment of endpoints used in clinical trialswhen assessing the efficacy of novel CSC therapies.These endpoints are currently based on tumour volumewhich may not correlate with CSC frequency. Novelclinical tests need to be developed in order to addressthis issue and be able to measure CSC frequency as wellas tumour volume.

A clinical test must have a high sensitivity and spec-ificity, be acceptable to the patient, logistically feasible,quick to perform and cost-effective. As discussedabove, the gold standard methods for assessing breastCSC activity experimentally are in vivo tumour forma-tion and serial transplantation assays. These methods

however are technically challenging, lengthy, expensive,have ethical implications and would be impractical toperform in a clinical trial setting.

Alternative in vitro methods such as colony-formingassays and identification of cell surface markers such asCD44+/CD24 have been utilised in pre-clinical studiesas surrogate measures of breast CSC function and expres-sion, respectively. In the clinical setting these methodshave been used in pre-surgical window trials to assessthe efficacy of treatments on the breast CSCs beforeand after treatment.21,22 However the technical expertise,

time and expense involved in performing these assayslimits their utility on a large scale. As an alternative to cellsorting, breast CSC markers could be used to detectCSCs using immunohistochemistry. In a series of 577breast cancer patients, Ginestier et al. (2007) demon-strated a correlation between ALDH1 expression andpoor prognosis.20 Whether this is due to increased num-ber or activity of breast CSCs remains unknown. Conse-quently before such immunohistochemical markers canbe used as surrogate measures of breast CSC expressionin clinical trials, they first need to be correlated with func-tional assays of CSC activity. The clinical utility of thismarker as well as other putative breast CSC markers suchas CD44 and CD24 to identify and monitor breast CSCsusing immunohistochemical techniques is further compli-cated by the heterogeneous nature of the disease as dem-onstrated by Park et al. (2010).96

An alternative approach to monitor breast CSCs isgene expression profiling using microarrays or RT-PCR(seeFig. 3). This could be applied to CSCs isolated from

primary tissue in neodjuvant trials or disseminatedtumour tissue/fluid in adjuvant trials. Several authorshave generated gene expression signatures from breastCSCs derived from primary breast cancer tissue andmammospheres which could be used to measure changesin expression of the breast CSC fraction from patients inresponse to treatment.23,97,98 Using a gene expressionsignature derived from human normal mammary stemcells, Pece et al. demonstrated that poorly differentiatedcancers displayed a higher content of CSCs.98 AlthoughPece et al. demonstrated that high grade tumours wereassociated with greater mammosphere formation and

tumour initiationin vivocompared to low grade tumoursin a small number of patient samples, the validity of thisand other gene signatures is limited by the lack of robustcorrelation with functional assays of CSC activity. Fur-thermore, it is possible that breast CSC gene signaturescould change as a result of complexgenetic and epigeneticchanges as the disease progresses, thus making monitor-ing using single gene expression profiles unreliable.

Tumour-derived epithelial cells or circulating tumourcells (CTCs) have been identified in the blood of breastcancer patients and are probably a mixture of breastCSCs and non-CSCs. The number of CTCs is an inde-pendent predictor of progression-free survival and over-

all survival in patients with metastatic breast cancer.99

Circulating breast CSCs are arguably the origin of meta-static disease and it would therefore be valuable if wecould monitor the frequency of these cells through geneexpression profiles or immunohistochemical markers inresponse to treatment. However these cells are extremelyrare which makes performing such tests technically chal-lenging. Furthermore, current methods of detectingCTCs utilise markers of epithelial cells whereas breastCSCs have a more mesenchymal phenotype and so maygo undetected.



10/13

CSCFrequency

TumourVolume

DR

DD

CSC treatment efficacy

PD Progressive Disease

SD Stable Disease

PR Partial Response

CR Complete Response

Traditional disease response

Potential disease responses with CSC

therapy

Other possible disease responses

Therapy

Traditional trial endpoints CSC treatment effect 1:

delayed relapse

CSC treatment effect 2:

dormant disease

CSC treatment effect 3:

complete cure

DR Delayed Relapse

DD Dormant Disease

CC Complete Cure

Detectable

tumour limit

DR

DD

CC

TherapyTherapyTherapy

CC

PD

SD

PR

CR

(a) (b) (c) (d)

Fig. 2. The predicted effects of novel cancer stem cell (CSC) therapies on tumour volume and CSC frequency (red) compared with the effect ofcurrent therapy alone (black). Current trials define endpoints based on tumour volumes which may not reflect the continual increase in CSCfrequency (a). To examine the efficacy of novel CSC treatments, CSC frequency as well as tumour volume must be assessed. CSC agents should becombined with current therapy to maximise impact on the tumour. As efficacy of the CSC agent increases, response to this combined therapyimproves from delayed relapse (b) to dormant disease (c) with complete cure (d) the most favourable outcome. (bd; comparing the combination ofnovel CSC agents with current therapy versus therapy alone which causes a complete response). (For interpretation of the references to colour in

this figure legend, the reader is referred to the web version of this article.)

Blood Samples

Identify CSC

population

Gene profiling

CSC markers

In vitro:

In vivo:

IHC FACS

Microarray RT-PCR

Tumourformation

Serialtransplantation

Colonyformation

3D

2D

Advanced cancer trial

Tissue Biopsies

Neoadjuvant trialCSC Expression

CSC Function

Fig. 3. Future clinical endpoints for novel cancer stem cell (CSC) therapies. To assess the efficacy of drugs targeting CSCs, CSC frequency shouldbe monitored. Ideally, the CSC population must be identified and its function both in vitro and in vivo should be assessed. CSCs from tumourbiopsies or isolated circulating tumour cells (CTCs) can be identified using CSC markers or by testing for a CSC gene expression profile (middlepanel). To functionally validate that the isolated cells are CSCs, assays to measure CSC activity would have to be performed (right handsidepanels).



11/13

7. Summary and conclusions

Breast cancer is a heterogeneous disease, and as yet,there are no universal molecular, cellular or geneticmarkers to isolate breast CSCs. Breast CSC markersare therefore not yet well-developed for their use in clin-ical trials. However, rapid advances in gene expressionprofiles and other technologies show great promiseand hopefully these can be translated into reliable, prac-tical and cost-effective methods which can be utilised inthe near future. The important question to be answeredwill not be are CSCs ready for the clinic, but becomes: isthe clinic ready for breast cancer stem cells? If we arerobust in our screens, carry out good validation inpre-clinical models and are careful in translation of thisconcept to clinical trials, we might then be able todevelop and deliver a CSC strategy to guide others inthe future.

Conflict of interest statement

None declared.

Acknowledgements

We wish to thank Anthony Howell, Frances Shaw,Gillian Farnie and Sacha Howell for discussion andfor carefully reading and commenting on the manu-script. M.P.A. is funded by BBSRC, J.K.S. is fundedby the Royal College of Surgeons of England andRBC is a Breast Cancer Campaign-funded Fellow.

References

1. Clarke MF, Dick JE, Dirks PB, et al. Cancer stem cells perspectives on current status and future directions: AACRWorkshop on cancer stem cells. Cancer Res2006;66(19):933944.

2. Bonnet D, Dick JE. Human acute myeloid leukemia is organizedas a hierarchy that originates from a primitive hematopoietic cell.Nat Med1997;3(7):7307.

3. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ,Clarke MF. Prospective identification of tumorigenic breastcancer cells. Proc Nat Acad Sci USA 2003;100(7):39838.

4. Wicha MS, Liu S, Dontu G. Cancer stem cells: an old idea aparadigm shift. Cancer Res2006;66(4):188390 [discussion 18951896].

5. Fillmore CM, Kuperwasser C. Human breast cancer cell lines

contain stem-like cells that self-renew, give rise to phenotypicallydiverse progeny and survive chemotherapy. Breast Cancer Res2008;10(2):R25.

6. Polyak K. Breast cancer stem cells: a case of mistaken identity?

Stem Cell Rev 2007;3(2):1079.7. Nowell PC. The clonal evolution of tumor cell populations.

Science 1976;194(4260):238.8. Hill RP. Identifying cancer stem cells in solid tumors: case not

proven. Cancer Res2006;66(4):18915 [discussion 1890].9. Adams JM, Kelly PN, Dakic A, Nutt SL, Strasser A. Response

to comment on tumor growth need not be driven by rare cancerstem cells. Science2007;318:1722d.

10. Quintana E, Shackleton M, Sabel MS, et al. Efficient tumour formationby single human melanoma cells.Nature2008;456(7222):5938.

11. Dontu G, Al-Hajj M, Abdallah WM, Clarke MF, Wicha MS.Stem cells in normal breast development and breast cancer. CellProlif2003;36(Suppl. 1):5972.

12. Ponti D, Costa A, Zaffaroni N, et al. Isolation and in vitropropagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res2005;65(13):550611.

13. Phillips TM, McBride WH, Pajonk F. The response of CD24(/low)/CD44+ breast cancer-initiating cells to radiation. J NatlCancer Inst 2006;98(24):177785.

14. Sheridan C, Kishimoto H, Fuchs RK, et al. CD44+/CD24breast cancer cells exhibit enhanced invasive properties: anearly step necessary for metastasis. Breast Cancer Res2006;8(5):R59.

15. Bauerschmitz GJ, Ranki T, Kangasniemi L, et al. Tissue-specificpromoters active in CD44+CD24/low breast cancer cells.

Cancer Res2008;68(14):55339.16. Honeth G, Bendahl PO, Ringner M, et al. The CD44+/CD24

phenotype is enriched in basal-like breast tumors.Breast CancerRes2008;10(3):R53.

17. Keller PJ, Lin AF, Arendt LM, et al. Mapping the cellular andmolecular heterogeneity of normal and malignant breast tissuesand cultured cell lines. Breast Cancer Res 2010;12(5):R87.

18. Harrison H, Farnie G, Howell SJ, et al. Regulation of breastcancer stem cell activity by signaling through the Notch4

receptor. Cancer Res2010;70(2):70918.19. Howell A, Wardley AM. Overview of the impact of conventional

systemic therapies on breast cancer. Endocr Relat Cancer2005;12(Suppl. 1):S9S16.

20. Ginestier C, Hur MH, Charafe-Jauffret E, et al. ALDH1 is amarker of normal and malignant human mammary stem cells anda predictor of poor clinical outcome. Cell Stem Cell2007;1(5):55567.

21. Yu F, Yao H, Zhu P, et al. Let-7 regulates self renewal andtumorigenicity of breast cancer cells. Cell2007;131(6):110923.

22. Li X, Lewis MT, Huang J, et al. Intrinsic resistance oftumorigenic breast cancer cells to chemotherapy. J Natl CancerInst 2008;100(9):6729.

23. Creighton CJ, Li X, Landis M, et al. Residual breast cancers afterconventional therapy display mesenchymal as well as tumor-

initiating features.Proc Nat Acad Sci USA2009;106(33):138205.24. Chuthapisith S, Eremin J, El-Sheemey M, Eremin O. Breast

cancer chemoresistance: emerging importance of cancer stemcells. Surg Oncol2010;19(1):2732.

25. Diehn M, Cho RW, Lobo NA, et al. Association of reactiveoxygen species levels and radioresistance in cancer stem cells.Nature2009;458(7239):7803.

26. Woodward WA, Chen MS, Behbod F, et al. WNT/beta-cateninmediates radiation resistance of mouse mammary progenitorcells. Proc Nat Acad Sci USA2007;104(2):61823.

27. Atkinson RL, Zhang M, Diagaradjane P, et al. Thermalenhancement with optically activated gold nanoshells sensitizesbreast cancer stem cells to radiation therapy. Sci Transl Med2010;2(55):55ra79.

28. Asselin-Labat ML, Shackleton M, Stingl J, et al. Steroid

hormone receptor status of mouse mammary stem cells. J NatlCancer Inst2006;98(14):10114.

29. Sleeman KE, Kendrick H, Robertson D, et al. Dissociation ofestrogen receptor expression and in vivo stem cell activity in themammary gland. J Cell Biol2007;176(1):1926.

30. Clarke RB, Howell A, Potten CS, Anderson E. Dissociationbetween steroid receptor expression and cell proliferation in thehuman breast. Cancer Res1997;57(22):498791.

31. Horwitz KB, Dye WW, Harrell JC, Kabos P, Sartorius CA. Raresteroid receptor-negative basal-like tumorigenic cells in luminalsubtype human breast cancer xenografts.Proc Nat Acad Sci USA2008;105(15):57749.

32. Kabos P, Haughian JM, Wang X, et al. Cytokeratin 5 positivecells represent a steroid receptor negative and therapy resistant



12/13

subpopulation in luminal breast cancers.Breast Cancer Res Treat2010;128(1):4555.

33. McClelland RA, Barrow D, Madden TA, et al. Enhanced epidermalgrowth factor receptor signaling in MCF7 breast cancer cells afterlong-term culture in the presence of the pure antiestrogen ICI182,780 (Faslodex). Endocrinology2001;142(7):277688.

34. Knowlden JM, Hutcheson IR, Jones HE, et al. Elevated levels ofepidermal growth factor receptor/c-erbB2 heterodimers mediatean autocrine growth regulatory pathway in tamoxifen-resistant

MCF-7 cells. Endocrinology2003;144(3):103244.35. Wright C, Nicholson S, Angus B, et al. Relationship between c-

erbB-2 protein product expression and response to endocrinetherapy in advanced breast cancer. Br J Cancer1992;65(1):11821.

36. Farnie G, Clarke RB, Spence K, et al. Novel cell culturetechnique for primary ductal carcinoma in situ: role of Notch andepidermal growth factor receptor signaling pathways. J NatlCancer Inst2007;99(8):61627.

37. Zhou J, Zhang H, Gu P, et al. NF-kappaB pathway inhibitorspreferentially inhibit breast cancer stem-like cells. Breast CancerRes Treat2008;111(3):41927.

38. Korkaya H, Paulson A, Iovino F, Wicha MS. HER2 regulatesthe mammary stem/progenitor cell population driving tumori-genesis and invasion. Oncogene2008;27(47):612030.

39. Magnifico A, Albano L, Campaner S, et al. Tumor-initiating cellsof HER2-positive carcinoma cell lines express the highestoncoprotein levels and are sensitive to trastuzumab. Clin CancerRes2009;15(6):201021.

40. Paik S, Kim C, Wolmark N. HER2 status and benefit fromadjuvant trastuzumab in breast cancer. N Engl J Med 2008;358(13):140911.

41. Joensuu H, Kellokumpu-Lehtinen PL, Bono P, et al. Adjuvantdocetaxel or vinorelbine with or without trastuzumab for breastcancer.N Engl J Med2006;354(8):80920.

42. Romond EH, Perez EA, Bryant J, et al. Trastuzumab plusadjuvant chemotherapy for operable HER2-positive breast can-cer. N Engl J Med2005;353(16):167384.

43. Gupta PB, Fillmore CM, Jiang G, et al. Stochastic statetransitions give rise to phenotypic equilibrium in populations of

cancer cells. Cell2011;146(4):63344.44. Chaffer CL, Brueckmann I, Scheel C, et al. Normal and

neoplastic nonstem cells can spontaneously convert to a stem-like state. Proc Nat Acad Sci USA 2011;108(19):79505.

45. Dontu G, Jackson KW, McNicholas E, et al. Role of Notchsignaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res 2004;6(6):R60515.

46. Androutsellis-Theotokis A, Leker RR, Soldner F, et al. Notchsignalling regulates stem cell numbers in vitro and in vivo. Nature2006;442(7104):8236.

47. Al-Husaini H, Subramanyam D, Reedijk MJ, Sridhar SS. Notchsignaling pathway as a therapeutic target in breast cancer. MolCancer Ther2010;10(1):915.

48. Jiang J, Hui CC. Hedgehog signaling in development and cancer.

Dev Cell2008;15(6):80112.

49. Liu S, Dontu G, Mantle ID, et al. Hedgehog signaling and Bmi-1regulate self-renewal of normal and malignant human mammarystem cells. Cancer Res 2006;66(12):606371.

50. Lawson ND, Vogel AM, Weinstein BM. Sonic hedgehog andvascular endothelial growth factor act upstream of the Notchpathway during arterial endothelial differentiation. Dev Cell2002;3(1):12736.

51. Kakarala M, Brenner DE, Korkaya H, et al. Targeting breaststem cells with the cancer preventive compounds curcumin andpiperine. Breast Cancer Res Treat 2010;122(3):77785.

52. Guzman ML, Neering SJ, Upchurch D, et al. Nuclear factor-kappaB is constitutively activated in primitive human acutemyelogenous leukemia cells. Blood2001;98(8):23017.

53. Malaguarnera L, Pilastro MR, DiMarco R, et al. Cell death inhuman acute myelogenous leukemic cells induced by pyr-rolidinedithiocarbamate. Apoptosis2003;8(5):53945.

54. Larsson SC, Mantzoros CS, Wolk A. Diabetes mellitus and riskof breast cancer: a meta-analysis. Int J Cancer 2007;121(4):85662.

55. Evans JM, Donnelly LA, Emslie-Smith AM, Alessi DR, MorrisAD. Metformin and reduced risk of cancer in diabetic patients.BMJ2005;330(7503):13045.

56. Liu B, Fan Z, Edgerton SM, et al. Metformin induces uniquebiological and molecular responses in triple negative breastcancer cells. Cell Cycle 2009;8(13):203140.

57. Hirsch HA, Iliopoulos D, Tsichlis PN, Struhl K. Metforminselectively targets cancer stem cells, and acts together withchemotherapy to block tumor growth and prolong remission.

Cancer Res2009;69(19):750711.58. Vazquez-Martin A, Oliveras-Ferraros C, Barco SD, Martin-

Castillo B, Menendez JA. The anti-diabetic drug metforminsuppresses self-renewal and proliferation of trastuzumab-resis-tant tumor-initiating breast cancer stem cells. Breast Cancer ResTreat2010;126(2):35564.

59. Thompson AM, Iwamoto T, Jordan L, Purdie C, Bray SE, BakerL, et al. Final analysis of the NEOMET trial of neoadjuvantmetformin: examining effects on Ki67, gene expression, and

pathway analysis in primary operable breast cancer. In: 2011ASCO Annual Meeting.J Clin Oncol2011;29:2011 (Suppl.; abstr534).

60. Ginestier C, Wicinski J, Cervera N, et al. Retinoid signalingregulates breast cancer stem cell differentiation. Cell Cycle2009;8(20):3297302.

61. Budd GT, Adamson PC, Gupta M, et al. Phase I/II trial of all-trans retinoic acid and tamoxifen in patients with advancedbreast cancer. Clin Cancer Res 1998;4(3):63542.

62. Altucci L, Gronemeyer H. The promise of retinoids to fightagainst cancer. Nat Rev Cancer 2001;1(3):18193.

63. Lawrence JA, Adamson PC, Caruso R, et al. Phase I clinical trialof alitretinoin and tamoxifen in breast cancer patients: toxicity,pharmacokinetic, and biomarker evaluations. J Clin Oncol2001;19(10):275463.

64. Zhou S, Schuetz JD, Bunting KD, et al. The ABC transporterBcrp1/ABCG2 is expressed in a wide variety of stem cells and is amolecular determinant of the side-population phenotype. NatMed2001;7(9):102834.

65. Patrawala L, Calhoun T, Schneider-Broussard R, et al. Sidepopulation is enriched in tumorigenic, stem-like cancer cells,whereas ABCG2+ and ABCG2 cancer cells are similarlytumorigenic. Cancer Res2005;65(14):620719.

66. Zhong Y, Zhou C, Ma W, et al. Most MCF7 and SK-OV3 cellswere deprived of their stem nature by Hoechst 33342. BiochemBiophys Res Commun 2007;364(2):33843.

67. Schinkel AH, Smit JJ, van Tellingen O, et al. Disruption of themouse mdr1a P-glycoprotein gene leads to a deficiency in theblood-brain barrier and to increased sensitivity to drugs. Cell1994;77(4):491502.

68. Cooray HC, Blackmore CG, Maskell L, Barrand MA. Locali-sation of breast cancer resistance protein in microvessel endo-thelium of human brain. Neuroreport2002;13(16):205963.

69. An Y, Ongkeko WM. ABCG2: the key to chemoresistance incancer stem cells? Expert Opin Drug Metab Toxicol2009;5(12):152942.

70. Hittelman WN, Liao Y, Wang L, Milas L. Are cancer stem cellsradioresistant? Future Oncol2010;6(10):156376.

71. Karimi-Busheri F, Rasouli-Nia A, Mackey JR, Weinfeld M.Senescence evasion by MCF-7 human breast tumor-initiatingcells. Breast Cancer Res 2010;12(3):R31.

72. Tutt A, Robson M, Garber JE, Domchek S, Audeh MW, WeitzelJN, et al. Phase II trial of the oral PARP inhibitor olaparib in



13/13

BRCA-deficient advanced breast cancer. In: 2009 ASCO AnnualMeeting. J Clin Oncol2009;27:18s (Suppl.; abstr CRA501).

73. Calvert H, Azzariti A. The clinical development of inhibitorsof poly(ADP-ribose) polymerase. Ann Oncol 2009;22(Suppl. 1):i539.

74. OShaughnessy J, Osborne C, Pippen JE, et al. Iniparib pluschemotherapy in metastatic triple-negative breast cancer. N EnglJ Med2011;364(3):20514.

75. Domagala P, Lubinski J, Domagala W. Iniparib in metastatic

triple-negative breast cancer. N Engl J Med 2011;364(18):1780[author reply 1781].

76. Monks NR, Biswas DK, Pardee AB. Blocking anti-apoptosis as astrategy for cancer chemotherapy: NF-kappaB as a target. J CellBiochem 2004;92(4):64650.

77. Madjd Z, Mehrjerdi AZ, Sharifi AM, et al. CD44+ cancer cellsexpress higher levels of the anti-apoptotic protein Bcl-2 in breasttumours. Cancer Immun2009;9:4.

78. Rom J, von Minckwitz G, Eiermann W, et al. Oblimersencombined with docetaxel, adriamycin and cyclophosphamide asneo-adjuvant systemic treatment in primary breast cancer: finalresults of a multicentric phase I study. Ann Oncol2008;19(10):1698705.

79. Rom J, von Minckwitz G, Marme F, et al. Phase I study ofapoptosis gene modulation with oblimersen within preoperative

chemotherapy in patients with primary breast cancer. Ann Oncol2009;20(11):182935.

80. Miyake K, Medina KL, Hayashi S, et al. Monoclonal antibodiesto Pgp-1/CD44 block lympho-hemopoiesis in long-term bonemarrow cultures. J Exp Med1990;171(2):47788.

81. Marangoni E, Lecomte N, Durand L, et al. CD44 targeting reducestumour growth and prevents post-chemotherapy relapse of humanbreast cancers xenografts. Br J Cancer2009;100(6):91822.

82. Guan JL. Integrin signaling through FAK in the regulation ofmammary stem cellsand breastcancer. IUBMB Life 2010;62(4):26876.

83. Owens LV, Xu L, Craven RJ, et al. Overexpression of the focaladhesion kinase (p125FAK) in invasive human tumors. CancerRes1995;55(13):27525.

84. Lightfoot Jr HM, Lark A, Livasy CA, et al. Upregulation offocal adhesion kinase (FAK) expression in ductal carcinoma

in situ (DCIS) is an early event in breast tumorigenesis. BreastCancer Res Treat2004;88(2):10916.

85. Luo M, Fan H, Nagy T, et al. Mammary epithelial-specificablation of the focal adhesion kinase suppresses mammarytumorigenesis by affecting mammary cancer stem/progenitorcells. Cancer Res2009;69(2):46674.

86. Provenzano PP, Inman DR, Eliceiri KW, Beggs HE, Keely PJ.Mammary epithelial-specific disruption of focal adhesion kinaseretards tumor formation and metastasis in a transgenic mousemodel of human breast cancer. Am J Pathol2008;173(5):155165.

87. Lahlou H, Sanguin-Gendreau V, Zuo D, et al. Mammaryepithelial-specific disruption of the focal adhesion kinase blocksmammary tumor progression. Proc Nat Acad Sci USA2007;104(51):203027.

88. Nagy T, Wei H, Shen TL, et al. Mammary epithelial-specific

deletion of the focal adhesion kinase gene leads to severe lobulo-alveolar hypoplasia and secretory immaturity of the murinemammary gland. J Biol Chem 2007;282(43):3176676.

89. Schultze A, Fiedler W. Therapeutic potential and limitations ofnew FAK inhibitors in the treatment of cancer. Expert OpinInvest Drugs2010;19(6):77788.

90. Charafe-Jauffret E, Ginestier C, Iovino F, et al. Breast cancer celllines contain functional cancer stem cells with metastatic capacityand a distinct molecular signature. Cancer Res 2009;69(4):130213.

91. Huang M, Li Y, Zhang H, Nan F. Breast cancer stromalfibroblasts promote the generation of CD44+CD24 cellsthrough SDF-1/CXCR4 interaction. J Exp Clin Cancer Res2010;29:80.

92. Iliopoulos D, Hirsch HA, Wang G, Struhl K. Inducible forma-

tion of breast cancer stem cells and their dynamic equilibriumwith non-stem cancer cells via IL6 secretion. Proc Nat Acad SciUSA2011;108(4):1397402.

93. Wicha MS. Regulation of breast cancer stem cells by cytokinenetworks. Mol. Targets Cancer Ther.2011;10(11 Suppl. 1).

94. Ginestier C, Liu S, Diebel ME, et al. CXCR1 blockade selectivelytargets human breast cancer stem cells in vitro and in xenografts.J Clin Invest 2010;120(2):48597.

95. Liu S, Ginestier C, Ou SJ, et al. Breast cancer stem cells areregulated by mesenchymal stem cells through cytokine networks.

Cancer Res2011;71(2):61424.96. Park SY, Lee HE, Li H, et al. Heterogeneity for stem cell-related

markers according to tumor subtype and histologic stage inbreast cancer. Clin Cancer Res 2010;16(3):87687.

97. Liu R, Wang X, Chen GY, et al. The prognostic role of a gene

signature from tumorigenic breast-cancer cells. N Engl J Med2007;356(3):21726.

98. Pece S, Tosoni D, Confalonieri S, et al. Biological and molecularheterogeneity of breast cancers correlates with their cancer stemcell content. Cell2010;140(1):6273.

99. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells,disease progression, and survival in metastatic breast cancer. NEngl J Med2004;351(8):78191.

100. Wright MH, Calcagno AM, Salcido CD, et al. Brca1 breasttumors contain distinct CD44+/CD24 and CD133+ cells withcancer stem cell characteristics. Breast Cancer Res2008;10(1):R10.

101. Cariati M, Naderi A, Brown JP, et al. Alpha-6 integrin isnecessary for the tumourigenicity of a stem cell-like subpopula-tion within the MCF7 breast cancer cell line. Int J Cancer

2008;122(2):298304.102. Meyer MJ, Fleming JM, Lin AF, et al. CD44posCD49fhiCD133/

2hi defines xenograft-initiating cells in estrogen receptor-negativebreast cancer. Cancer Res2010;70(11):462433.

103. Calcagno AM, Salcido CD, Gillet JP, et al. Prolonged drugselection of breast cancer cells and enrichment of cancer stem cellcharacteristics. J Natl Cancer Inst 2010;102(21):163752.

104. Lagadec C, Vlashi E, Della Donna L, et al. Survival and self-renewing capacity of breast cancer initiating cells duringfractionated radiation treatment. Breast Cancer Res2010;12(1):R13.

105. Zhang M, Behbod F, Atkinson RL, et al. Identification of tumor-initiating cells in a p53-null mouse model of breast cancer. CancerRes2008;68(12):467482.

106. Hoey T, Yen WC, Axelrod F, et al. DLL4 blockade inhibits

tumor growth and reduces tumor-initiating cell frequency. CellStem Cell2009;5(2):16877.

107. Kubo M, Nakamura M, Tasaki A, et al. Hedgehog signalingpathway is a new therapeutic target for patients with breastcancer. Cancer Res 2004;64(17):60714.


Documents

Stem cells in breast tumours- Are they ready for the clinicď€Ą, 2012