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Review
Breast cancer stem cells: an update
  1. Jabed Iqbal,
  2. Pek Yoon Chong,
  3. Puay Hoon Tan
  1. Department of Pathology, Singapore General Hospital, Singapore, Singapore
  1. Correspondence to Dr Jabed Iqbal, Department of Pathology, Singapore General Hospital, One Hospital Drive, Singapore 169608, Singapore; jabed.iqbal{at}sgh.com.sg

Abstract

Breast cancer is a significant cause of morbidity and mortality in women with a high incidence of recurrence or treatment failure. Growing evidence suggests that cancer stem cells (CSCs) most likely contribute to tumour progression, spread and therapy failure. However, despite extensive research and the tremendous clinical potential of such cells in possible therapeutic management, the real nature of CSCs remains an enigma. In this review, we discuss the fundamental properties and molecular target of CSCs and focus on recent advances regarding the identification of CSC markers with emphasis on breast cancer and the underlying molecular mechanism of CSC phenotypes. We also discuss experimental evidence of targeting molecular pathways in order to modulate breast CSC behaviour in tumourigenesis and the controversies associated with it that potentially weaken the CSC model in breast cancer and other cancers as well.

  • Breast Cancer
  • Cancer Stem Cells
  • Molecular Oncology

https://doi.org/10.1136/jclinpath-2012-201304

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    Introduction

    Breast tumour cells which display properties innate to stem cells, that is, tumour-initiation, self-replication and self-renewal, have been termed breast cancer stem cells (CSC).1–3 Substantial effort has been directed to identify biomarkers for better isolation, characterisation and targeting of CSCs. In this review, the general features of CSCs with emphasis on their characteristics in breast cancers as well as the controversies related to CSC are discussed.

    CSC origin

    Two likely sources have been proposed as the origin of CSCs: (a) stem cells or (b) committed progenitor cells stimulated by multiple mutational events.4 A third pathway has also been suggested whereby CSCs originate from mature somatic cells.

    • Hypothesis #1: Stem cells give rise to cancer cells, distinguished by: (a) self-renewal and (b) pleuripotency using the existing stem cell regulatory pathways to promote self-renewal and therefore long lifespan.5

    • Hypothesis #2: Progenitor cells give rise to cancer cells via an intermediate cell type involved in the transformation from a stem cell to a differentiated form. The division of latter form results in mature cells with partial ability of self-renewal.6 ,7

    • Hypothesis #3: Mature, differentiated cells give rise to cancer cells that de-differentiate to possess stem cell-like phenotype and assume self-renewal properties.8 ,9

    Breast cancer CSC

    The first solid CSCs were identified in breast tumours in 2003; subsequently, CSCs have been described in colonic, pancreatic, prostatic, ovarian, hepatic, lung, gastric, melanoma and brain tumours cancers.10 ,11 A subpopulation of lineage-negative cancer cells with increased expression of membranous CD44 and absent or reduced levels of CD24 (CD44hiCD24lo) was isolated by Al-Hajj and his coworkers; this population had enhanced tumour-forming capability compared with bulk tumour cells.11 Subsequent studies have shown that CD44hiCD24lo cells have a greater capacity to propagate as mammospheres, which is an in vitro surrogate assay for self-renewal.2 Also, a higher proportion of CD44hiCD24lo cells in breast cancer have been associated with shorter recurrence-free and overall survival with increased distant metastases.2

    CSC biomarkers

    See table 1.

    View this table:
    Table 1

    Biomarkers in breast cancer stem cells

    Side population

    Side population (SP) cells with stem cell-like properties have been identified in a variety of human haematological and solid malignant cells following identification using dual wavelength flow cytometry combined with Hoechst 33 342 dye efflux.12 In addition to displaying CSC phenotype, these cells also have an enhanced rate of Hoechst efflux attributed to the expression of members of the ABC transporter family. However, recent studies have identified problems in using SP cells as a CSC fraction because of conflicting results due to cross-contamination of the SP and non-SP fractions.13

    CD44

    This is a useful marker for identifying CSCs in breast tumours as well as in a variety of other tumours.11 ,14 Implantation of patient tumours or breast CSCs into mouse mammary fat pads complemented by the use of non-invasive imaging strategies has demonstrated high tumourigenicity of CD44 cells from both primary tumours and lung metastases.14

    CD133

    Recent studies have demonstrated that CD133 (prominin-1) is a specific marker of CSCs in a wide spectrum of cancers.15 Brca1 breast tumours have been shown to contain distinct CD133 as well as CD44/CD24− cells which exhibit CSC characteristics.16 Similarly, CD133 cells with CSC phenotype have been identified in triple-negative breast cancers.17

    CD44/CD24

    Although CD44+CD24−/low population appears to enrich for CSCs, expression of stem cell markers, including CD44+CD24−/low and ALDH1A1, varied between both breast cancer cell lines and in vivo primary tumours. In fact, these markers do not consistently enrich for CSCs.9 This phenotypic heterogeneity of breast CSCs is not unique and has also been observed in haematological malignancies where multiple clones of tumour cells have been described.10 ,11

    ALDH activity

    ALDH1 is a detoxifying enzyme responsible for the oxidation of intracellular aldehydes. In one study, ALDH-bri cells constituted a significant proportion of epithelial cells in invasive ductal carcinomas, were greatly enriched for tumourigenic capacity and displayed some overlap with the CD44CD24− fraction.18 ,19 Interestingly, presence of ALDH1-positive cells is higher in basal-like and HER2-positive tumours compared with luminal subtypes.20 However, ALDH activity does not universally select for the most clonogenic cells in certain breast cancer cell lines.21

    Other biomarkers

    Gene-expression profiling of human breast stem/progenitor cells has revealed high levels of biomarkers including progesterone receptor (PR) and Notch 4 in myoepithelial cells. Low levels of estrogen receptor (ER) and Notch 3 were identified in the bipotent human mammary epithelial progeni­tor cells which are predominantly CD44.22–25 In contrast, the committed luminal progenitor cells showed the reverse phenotype, that is, high levels of ER and Notch 3 accompanied by low levels of PR and Notch 4 in luminal epithelial cells. Interestingly, endothelial cell protein C/activated protein C (EPCR/PROCR) appears to be associated with basal-like tumours.23

    Gene signatures in breast CSCs

    A series of breast CSC signatures has been identified and associated with poor prognosis. Patients with a high expression of CD44 signature genes exhibited shorter metastasis-free survival than patients with an increased CD24 signature gene expression, which appeared to be independ­ent of ER status and tumour grade.23 An invasiveness signature predicting increased risk of death and metastasis in ER-positive and grade 2 tumours was demonstrated by gene-expression profiling of CD44+/CD24−/low breast cancer cells.26 A CD44+/CD24−/low mammosphere-forming gene signature was associated with the claudin-low molecular subtype of breast cancer, which displayed epithelial–mesenchymal transition (EMT) marker enrichment and resistance to both hormone and chemotherapy, was identified by Creighton et al.27 Embryonic stem cell signatures have also been correlated with poor survival outcome.28 The marked diversity observed among these gene signatures in CSCs warrants further studies to validate and prognosticate their expression as CSC biomarkers.

    miRNA and breast CSC

    MicroRNAs (miRNAs) are small non-coding regulatory RNAs that regulate the translation of mRNAs by inhibiting ribosome function, decapping the 5′-Cap structure, deadenylating the poly(A) tail and degrading the target mRNA. The recent identification of miRNAs as important players in breast cancer has led to exploration of the molecular mechanism underlying breast CSC phenotypes. Shimono et al found a set of 37 miRNAs that were differentially expressed between human breast CSCs and lineage non-tumourigenic breast cancer cells.29 Three clusters, miR-200c-141, miR-200b-200a-429 and miR-183-96-182, were downregulated in breast CSCs, normal human and murine mammary stem/progenitor cells, and embryonal carcinoma cells.

    miR-200c strongly suppresses the ability of normal mammary stem cells to form mammary ducts and tumour formation driven by human breast CSCs in vivo.29 Let-7, another miRNA, suppresses self-renewal in breast CSC and regulates breast CSC phenotypes including chemoresistance and asymmetric cell division.30 ,31

    Signalling pathways modulating breast CSC

    Several signalling pathways, particularly Hedgehog (Hh), Wnt/β-catenin and Notch signalling systems, play a role in embryogenesis and organogenesis as well as in maintenance of tissues in the adult through regulation of the balance between self-renewal and differentiation of stem cells (table 2). In the mammary gland, these three signalling pathways play a significant role in stem cell self-renewal. Potential deregulation of these CSC-specific pathways may lead to neoplasia. On the other hand, these represent potential targets for therapy for breast CSCs.

    View this table:
    Table 2

    Cell signalling pathways in breast cancer stem cells

    The Notch transmembrane signalling proteins are expressed in both stem cells and early progenitor cells.32 Overexpression of Notch-4 has been shown to suppress (a) differentiation of breast epithelial cells in vitro and (b) development of normal mammary glands while promoting the development of mammary tumours in vivo.33 ,34 These observations suggest that modulation of Notch-4 signalling is important in the transformation of normal mammary stem cell to a CSC.

    The Wnt pathway is involved in cell fate determination in many organs including the developing mammary gland; overexpression of Wnt in mouse mammary glands can lead to increased mammary tumour formation.35 ,36 The pro-oncogenic role of β-catenin, a downstream target of Wnt signalling, has also been described in breast cancer. Taken together, these data indicate the involvement of Wnt signalling pathway members including β-catenin in the deregulation and transformation of stem cells into CSCs.

    The Hh/Patched pathway is important for embryonic growth and cell fate determination during development. The PITCH membrane protein (product of the tumour suppressor gene Patched) is a receptor for the Hh family of signalling molecules and has been implicated in early embryonic tumourigenesis.37 Importance of this pathway is signalled by its involvement in several types of cancer including breast, prostate and lung cancer.38

    Recently published data indicate that EGFR signalling is required for cancer cell self-renewal in mammospheres which in turn alludes to the susceptibility of these tumourigenic cells to EGFR-specific chemotherapeutic agents in vivo.39 ,40 In line with these findings, lapatinib led to a decrease in the percentage of tumourigenic cells in matched biopsies, although it was not statistically significant.39

    EMT and breast SCS

    The Weinberg group has recently reported that induction of EMT in epithelial cells led to acquisition of stem cell properties.41 ,42 Moreover, EMT has been shown to be induced in immortalised human mammary epithelial cells either by treatment with TGF-β or by exogenous expression of the proteins Snail or Twist resulting in the acquisition of the CD44hiCD24lo profile as well as significantly greater mammosphere formation.43 These results imply that EMT may be linked to the CSC ‘state’ whereby tumour cells are induced to acquire stem cell-like properties by EMT thereby becoming more tumourigenic and invasive.

    Cancer propagation models

    While most neoplasms originate from single cells, tumour progression is initiated and propagated by acquisition of genetic modifications within the original clone resulting in subsequent selection of more aggressive clones.44–47 Two alternative models to explain progression of solid cancer have been proposed: (a) the clonal evolution model which assumes the homogeneity of tumour whereby every tumour cell is potentially tumour-initiating, governed by low-probability random events. (b) The CSC model which states that the tumour is functionally heterogeneous and a subset of tumour cells with stem cell-like properties drive tumour-initiation, progression and recurrence/metastasis.47 ,48 However, neither model has been proven to be exclusive in nature. The likely explanation is that tumours may follow either pathway or may exhibit features of a mixed growth model. A more interesting model to explain the nature of sustained tumour growth has been proposed based on the pre-existing CSC model. According to this hypothesis, tumours could originally be driven by a subset of CSCs. Subsequent mutations imparting self-renewal leads to a more aggressive clone phenotypically distinct from the original CSC. However, if the subclone lacks these ‘stem cell-like’ properties, it may not be able to initiate tumours with a high frequency.48

    Identification of CSCs

    1. Immunohistochemistry has been used as the principal modality for detection of putative stem cell-associated biomarkers due to easy availability of paraffin-embedded tissue tissues. CD44, CD24 and ALDH1 expression coupled with cytokeratin detection has been performed in human breast cancers by immunohistochemical methods (figure 1A–C). However, results have been variable and occasionally contradictory.49–52

    2. Fluorescent-activated cell sorting (FACS) analysis has been used as the principal method for in vitro separation of stem and non-stem cells.53 Two categories of markers are used for cell sorting: (a) Cell surface markers: CD44/CD24−/low, elevated ALDH1+, CD24high/CD49Fhigh/Delta-notch-like EGF (epidermal growth factor) repeat-containing transmembrane (DNER)high, CD24high/CD49Fhigh/Delta-like1(DLL1)high, etc.23 ,54–57 (b) Cytoplasmic markers: Detection of aldehyde dehydrogenase has been performed using mammosphere cultures coupled with the ALDEFLUOR assay, the latter measuring cell enzymatic activity.56–59 ALDH1 expression in inflammatory breast cancer cells has been shown to be associated with metastasis and poor outcome.60

    3. Xenotransplantation assays are used frequently to assess stem cell activity due to its similarity to in vivo tumour microenvironment. Using FACS analysis, a population of tumour-initiating cells are sorted and injected into the mammary fat pad of immunocompromised mice. Cells with stem cell function, even at low concentrations, can generate tumours.61–63 However, the procedure is limited by the inherent presence of small numbers of contaminating cells that precludes from isolating relatively pure stem cell populations by FACS analysis. Furthermore, the tumourigenesis depends on the background of the animal models.49

    4. Mammosphere assays (anchorage-independent culture) have been mostly used as the principal assay to measure the stem cell activity of a sorted cell population in vitro which allows mammary epithelial cell proliferation in an undifferentiated state as spherical mammospheres enriched in stem cells and progenitor cells.64 ,65 Several studies have demonstrated that the efficacy of mammosphere formation increased significantly following neoadjuvant chemotherapy.17 ,18 Interestingly, the addition of an EGFR inhibitor, lapatinib, to the neoadjuvant therapy inhibits mammosphere-forming efficiency.27 ,66 Thus, the development of mammosphere assays is important for both studying the biology of breast CSCs and understanding the underlying key molecular pathways in both normal and neoplastic stem cells.

    Figure 1
    Figure 1

    Immunohistochemical staining of invasive ductal carcinoma for CD44 and CD24. CD44 is stained with Permanent Red and CD24 with diaminobenzidene. Magnification (A) A tumour showing predominantly membranous CD44 stain, 20×. (B) A tumour showing membranous and cytoplasmic CD24 stain, 20×. (C) A tumour with predominantly CD44/CD24− cells (arrow). A few CD44/CD24 cells are present showing weak brown cytoplasmic staining (circle), 40×.

    Inhibition of CSC

    Signalling pathways in CSCs including Wnt, notch and Hh signalling pathways represent an emerging new field of cancer therapeutics. Rizzo and coworkers found that oestrogen-inhibited notch activity was mediated in part through inhibition of γ-secretase activity suggesting that inhibition of notch signalling may be a useful strategy in breast cancer.67 Chen et al identified two new classes of small molecules that inhibit wnt signalling: (a) acyltransferase and (b) pro-axin.68 Multiple drugs active at different levels of the Wnt pathway (receptor–ligand interaction, cytosolic and nuclear signalling) are being developed or tested in clinical trials.69 ,70 Salinomycin, an antibiotic potassium ionophore, a selective breast CSC inhibitor, eradicates breast CSCs effectively in mouse models, but is relatively non-toxic to normal stem cells; evidence suggesting that salinomycin inhibits proximal Wnt/β-catenin signalling.71

    CSCs express breast cancer resistance protein (BRCP-ABCG2) and MDR-associated protein-1 (ABCB1/MDR1), the former conferring the SP phenotype and both being partially responsible for chemotherapeutic resistance.38 Molecules like UHRF1 which inhibit MDR1 transcription and expression have been proposed as CSC antagonists. Combining ABC transporter inhibitors and chemotherapy could be effective in eradicating CSC.72

    Targeting multiple signalling pathways interacting through cross-talk as part of broad, complex signalling networks has been a potential target for eradication of CSCs. For example, Her2-overexpressing breast cancer cell lines had several fold lower levels of notch transcriptional activity when compared with tumour cells expressing low levels of Her2, suggesting that Her2 overexpression suppresses notch signalling pathway.73 Treatment of these cells in vitro with gefitinib, an EGFR inhibitor, resulted in decreased expression of both EGFR and notch1. Moreover, Hh presumably modulates CSC tumourigenic property in cooperation with notch interaction indicating the important cross-talk between Hh signalling and notch in breast cancer.74

    Controversy

    The very existence of CSCs has been subjected to extensive controversy from the very beginning, but the premise of the CSC model is attractive because it provides a potential modality of curing cancer by eradicating CSC. However, sceptics have maintained that CSC does not necessarily lead to tumour formation as shown by Quintana et al. They postulated that, at least in melanoma, CSC does not exist as tumour-initiating cells.75 The problems of the current technology used to study CSC are multifactorial: (a) It has been argued that the transformed cell lines used are highly mutated populations and the phenotype does not realistically resemble that of primary cancer cells, (b) accurate techniques to determine self-renewal in order to identify CSC and (c) the role of microenvironment of the host stroma has mostly been overlooked in most experiments.76 In the absence of an identifiable ‘stem cell niche’, the role of nude mice as xenotransplantation hosts has been criticised by CSC sceptics.

    Multiple observations have suggested that CSCs may be associated with more aggressive subtypes of breast cancer. For example, CD44/CD24− cells are associated with basal-like and metaplastic cancers, ALDH1+ cells are more common in HER2-positive and basal-like tumours and claudin-low tumours associated with EMT have been related to CSC phenotype.77 These observations have led to some controversial issues in breast cancer: are CSCs in different molecular (intrinsic) subtypes of breast cancer similar or are these seen only in aggressive breast tumours as suggested by some authors? More importantly, do the limitations of the currently used assays invalidate the existence of CSCs?

    Conclusions

    In breast tumours, CSCs may constitute a subpopulation that persists following therapy which can explain the prevalence of cancer treatment failure. Presently there are neither methodologies to unequivocally identify CSCs nor validated treatment strategies to target CSCs. Hence, a few critical biological questions need to be addressed which, in addition to identifying the CSC, include: (a) how to differentiate between CSCs and normal adult stem cells, (b) which regulatory events control the emergence of CSCs within a tumour, (c) how the individual characteristics of tumour-specific CSCs differ between patients and (d) how the original genomic characteristics of a CSC clone dictate the histology and clinical behaviour of the tumour that emerges.78 Recently, independent studies using lineage-tracing technique in mouse models have successfully identified the existence of CSCs in brain, skin and intestinal tumours.79 ,80 Similarly, Spike et al have identified fetal mammary stem cells that express CD24 and CD44 and possess similarities to basal-like and Her2-positive breast cancers.81 These results may provide resources to identify ‘adult’ breast CSC in human or mouse models and thereby refine therapeutic strategies targeting these cells.

    Key messages

    • Breast tumour cells which display properties innate to stem cells, that is, tumour-initiation, self-replication and self-renewal, are called breast cancer stem cells.

    • Combination of biomarkers can be used to identify cancer stem cells in breast tumors

    • Dysregulation of stem cell-specific pathways lead to neoplasia. On the other hand, these pathways represent potential targets for breast cancer stem cell eradication.

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    Acknowledgments

    Acknowledge Dr Aye Aye Thike for providing the images.

    No patient data used

    Provided by Centralised Institutional Review Board, SingHealth

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    Footnotes

    • Contributors JI: conception and design, drafting the article or revising it critically for important intellectual content. AC and TPH: revising it critically for important intellectual content.

    • Competing interests None.

    • Provenance and peer review Not commissioned; externally peer reviewed.