review article by dr.Radiana D A,M.Biomed
Abstract
Identification of human breast cancer stem cells (BrCa SCs) changed the landscape of breast cancer treatment. Solid tumours such as breast cancers contain a small fraction of self-renewing tumourigenic cells that give rise to and maintain the bulk of the tumour mass. This review presents highlights on breast cancer stem cells and discusses strategies and limitations to accomplishing the ultimate goal of eradicating breast cancer stem cells.

Introduction
Breast cancer has been, and still is, one of the most aggressive female killers. In 2008, about 40,480 women will die from breast cancer in the United States.1 The chance of developing invasive breast cancer at some time in a woman's life is about one in eight (12%).1 The incidence of the disease has apparently increased throughout the world during the past century. Women living in North America have the highest rate of breast cancer in the world. Caucasian women in the Western world have a considerably higher breast cancer risk than Asian women in China or Japan.2 The incidence of breast cancer in Indonesia, according to Globocan data, IARC 2002, is 26 per 100,000 women.3
Although the theory of cancer stem cells dates back to more than 50 years ago,4 the existence of the first bona fide tumourigenic cell compartment was not demonstrated until 1994, when the acute myelogenous leukaemia stem cell (LSC) was identified and characterized.5 However, researchers doubted the existence of an analogous cell type in solid tumours. It was believed that every cell from a tumour had an equal likelihood of seeding a secondary cancer as long as it was in the correct microenvironment.
However, in 2003, an influential report describing the prospective identification of human BrCa SCs changed the landscape of breast cancer research.6 Most researchers now consider that solid tumours such as breast cancers contain a small fraction of self-renewing tumourigenic cells that give rise to and maintain the bulk of the tumour mass. Successfully targeting these cancer stem cells for eradication therefore requires a better understanding of how a good stem cell could go bad in the first place. This review presents highlights on the research of breast cancer stem cells, and discusses strategies for and limitations to accomplishing the ultimate goal of eradicating BrCa SCs.
Identification of Breast Cancer Stem Cells
Stem cell is defined as a cell with three distinct properties: self-renewal, the capability to develop into multiple lineages, and the potential to proliferate extensively. Despite the growing evidence of stem cell therapy for degenerative disease such as myocardial infarction or cerebrovascular accident, it is now known that stem cells exist in tumour bulk mass; they are the initiating cells that cause cancer and maintain tumour bulk mass. The paper by Al-Hajj et al provides a major step forward with the identification of human breast cancer initiating cells (BrCa SCs) through reliable xenograft assay that enabled the injection of single-cell suspensions from human breast cancer tissue into the mammary fat pad of genetically engineered immune-deficient mice.6 Breast cancer tissue obtained from nine samples (metastatic and primary breast tumours) was sorted by distinguished cell surface markers using a fluorescence activated cell sorting (FACS) machine. Fragments of breast cancer tissue containing haematopoetic, endothelial, mesothelial and fibroblast cell lineage marker (Lin+) were separated first from Lin− fragments. Al-Hajj et al then used four cell surface markers (adhesion molecules CD44 and CD24, a breast/ovarian cancer specific marker B38.1, and the epithelial-specific antigen [ESA]), whose expression is heterogeneous in breast cancer tissue, to sort into expressing and nonexpressing fractions, alone or in combination. The breast cancer initiating capacity of each cell population was determined by the mice xenotransplant assay.
All mice injected with either CD44+, B38.1+ or CD24−/low generated palpable tumours in 12 weeks, whereas none of the CD44− or B38.1− injections caused tumours. For three samples, CD44+CD24 /low fractions were subdivided by expression of ESA: ESA+ cells generated breast tumours in mice, whereas ESA cells did not. ESA+ represented a minor subpopulation (2%), and dilution analysis showed that breast cancer initiating activity was enriched by 50-fold in this fraction. Tumours in mice contained the same complex mixture of ESA , CD44− and CD24+ cells as tumours from the original donor. These results conclusively demonstrate that a rare breast cancer stem cell is the only cell type capable of establishing human breast cancer and re-establishing functionally heterogeneous breast cancer after transplantation into mice. Moreover, the BrCa SCs express unique cell surface markers that enable prospective isolation.7
At cell division, the BrCa SC can self-renew as well as make progeny that acquire maturation markers and lose the ability to initiate tumour growth. This process is very similar to the process whereby normal stem cells can both self-renew to maintain the stem cell pool and also produce differentiated progeny to create the mature cell types specific to that organ. Thus, breast cancer cells retain remnants of normal developmental programmes.7
Ponti and colleagues employed a similar approach to derive mammospheres from human breast cancers.8 Mammosphere cells were undifferentiated but, interestingly, a large majority had the same CD44+CD24− phenotype reported by Al-Hajj and colleagues. When transferred to differentiating culture conditions, mammospheres produced cells of both luminal and basal/myoepithelial lineages. Mammospheres initiated cancers in the cleared mammary fat pad of immunodeficient mice at 1,000-fold greater dilutions than established breast cancer–derived cell lines. Following enzymatic dissociation, 10% to 20% of cells from primary cancer–derived mammospheres formed secondary mammospheres, and several cultures were serially expanded for more than 40 passages, proving a capacity for long-term self-renewal. Mammospheres from more aggressive cancers formed a larger number of secondary mammospheres, suggesting a greater capacity for self-renewal.
The results of both Al-Hajj and Ponti suggest that breast cancer cells with the capability for long-term self-renewal are enriched within the CD44+CD24− subset. We should not, however, infer from this that the CD44+CD24− subset of cancer cells is a homogeneous population of cancer stem cells; instead, this population most probably represents a heterogeneous mix of cancer stem cells and early progenitor cells.9 Therefore, we will infer that the CD44+CD24− subset of cancer cells is a subset of putative BrCa SCs.
Several studies that have attempted to repeat and expand on the CD44+/CD24− putative BrCa SCs have thus far been inconclusive. In 2004, a clinical study reported that there was no statistically significant CD44 or CD24 staining in primary breast cancer sections in relation to tumour grade, type or size. The authors postulated that one difference is that they used primary sections, whereas the study by Al-Hajj et al used cell sorting to remove Lin− cells and subsequent flow cytometry. However, two more recent reports have now confirmed, both in breast cancer–derived cell lines and breast tumours, that CD44+/CD24− phenotypes are not necessarily associated with patient outcome or the ability to metastasize.10,11
In 2007, Shipitsin and coworkers found that CD24 is expressed on more differentiated cells whereas CD44 is expressed on more progenitor-like cells.12 Specifically, they found that breast cells of the CD44+/CD24− phenotype express genes that are involved in cell motility and angiogenesis, are more mesenchymal and motile, and are predominately oestrogen receptor negative. Breast cancer cells with a basal-like or mesenchymal-like phenotype are present in CD44+/CD24− cells. In contrast, cells with a more luminal, epithelial appearance (luminal differentiated mucin-1-positive, oestrogen receptor/progesterone receptor–positive, Gata3-positive cells) were largely CD44−/CD24+. This study suggests another interesting interpretation of CD24 and CD44 as markers of breast cancer cells; perhaps CD44+ cells are predominately basal-like and therefore are present in poor prognosis basal-like tumours, whereas CD24+ cells are luminal-like and therefore present in more differentiated luminal-type cancers.
Origin of Breast Cancer Stem Cells
One of the most important questions is whether BrCa SCs originate from normal adult mammary epithelial stem cells (MESCs) or from a transit-cell population in the normal breast. Breast cancer stem cells, and the cancer they generate, might have very different characteristics depending on which of these normal populations the BrCa SCs arise from — for instance, it could mean the difference between being poorly differentiated and highly aggressive, or relatively well differentiated and noninvasive.12
Some experimental evidence supports the hypothesis that normal stem cells are indeed the primary targets for tumourigenesis in the adult mammary gland, and form the cancer stem–cell population.13,14 There are several mechanisms that might explain the link between MESCs and the risk of neoplasia. Stem cells are thought to be long-lived and have a large replicative potential. This means that not only will they persist in the body long enough to accumulate the many mutations that are required to change a normal cell into one with neoplastic potential (a putative cancer stem cell), but they also have the proliferative capacity to actually generate a cancer mass. If these characteristics persist when normal stem cells progress to being putative cancer stem cells, they might be resistant to traditional chemotherapy and therefore have the ability to survive an initial cancer kill and generate cancer transit cells, which might allow the cancer to regrow once the chemotherapy regimen has ended.13
Stem cells might also explain ‘field cancerization’.15 This concept suggests that preneoplastic fields of cells might develop because of their clonal origin from an original cell with a mutation — for instance, the loss of a cancer-suppressor gene. Such a mutation would be phenotypically silent, but would predispose all cells in the field to neoplastic development, even if they were relatively short-lived (compared with stem cells). So, even a single mutation in a stem cell could generate a cancer-prone field, leading to apparently independent cancers arising from nearby sites. The importance of
this would relate to the rate of turnover of transit amplifying cells in the field — in other words, whether they are present long enough for additional mutations to arise. Cells in the preneoplastic field have an advantage over cells outside the field, but additional hits still need to occur. The most extreme case of field cancerization would be the inheritance of a germline mutation in a cancer suppressor gene, such as BRCA2 or TP53. In that case, the field comprises the whole mammary gland.

Transforming Factors to Breast Cancer Stem Cells
The putative BrCa SCs were driven by an activated molecular pathway that makes them resemble normal stem cells. Women whose breast tumours are largely made up of these ‘stem-like’ cells are at higher risk of recurrences. A study by Shipitsin showed that CD44+ cells were driven by the abnormal activated TFG-β1 (transmembrane receptor protein tyrosine kinase) pathway.12
There is increasing evidence that aberrant activation of the Wnt signalling pathway may result in the initiation of self-renewal of BrCA SCs.17 Caroline Alexander and colleagues showed that progenitor cells as a cell of origin for Wnt pathway–induced mammary tumourigenesis come from the identification of an increased mammary progenitor population, as assessed by the expression of Sca-1 and keratin 6, in both Wnt-1 mammary tumour virus–induced hyperplasias and tumours.17 Keratin 6 and Sca-1 appear to be expressed in mammary gland progenitor cells.16 Analysis for loss of heterozygosity of the tumour suppressor gene PTEN in hyperplasias and tumours present in mice strongly supported the hypothesis that a bipotential progenitor cell was the cell of origin.
Mammary tumours that arose in mice expressing the Neu, H-Ras or Polyoma middle T antigen transgenes driven by the MMTV promoter did not exhibit a similar increase in the Sca-1 and keratin 6-positive progenitor cell population.17 Furthermore, a recent study suggested that the MMTV-Neu tumours arose from parity-induced mammary epithelial cells, whereas MMTV-Wnt-1 tumours originated from ductal epithelial subtypes.17 These studies demonstrated that the enrichment of a particular cell population in a hyperplasia or tumour may be highly dependent on the type of initiating event as well as the cell type in which these events occur.
Aberrant Notch regulation may cause breast cancer.18 Transgenic mice expressing MMTV-Notch 4 develop mammary tumours within 4 to 6 months. The tumours in these mice were highly malignant and metastatic. It was speculated that the role of Notch 4 in tumourigenesis is dependent on the genetic background and the timing of Notch 4 expression with respect to the stage of mammary gland development. Whether activation of Notch 4 results in the expansion of specific mammary progenitors and induction of tumourigenesis remains to be established, but it appears likely. It is likely that the Notch pathway will exert pleiotropic effects on both stem cell self-renewal and differentiation. Recent support for this hypothesis has come from studies of Notch signalling in mammosphere cultures by Dontu et al.19
The Hedgehog (Hh) signalling pathway is another example of a pathway essential for development whose aberrant expression results in a variety of different human cancers, such as breast cancer and mammary ductal hyperplasias.20 However, the relation between the Hh signalling pathway and BrCa SCs has yet to be established.
The importance of the regulatory role of the mammary stroma and microenvironment in normal development as well as carcinogenesis has been known for many years. Kuperwasser et al have revisited this idea and shown that premalignant mammary epithelial cells adopt a malignant growth pattern when cultured with cancer-derived stromal cells or when transplanted to irradiated mammary fat pads, but not when cultured in the presence of normal mammary stromal cells or transplanted to nonirradiated fat pads.21 The precise mechanisms mediating cancer promotion in mammary stroma remain unclear; possibilities include alterations in expression of growth factors and matrix remodelling enzymes, the recruitment of inflammatory cells and the elaboration of viral oncoproteins.9
Possible Mechanism in Eradication of Breast Cancer Stem Cells
Whether stem cells themselves accumulate mutations to generate neoplasia, or whether they establish a clone of cancer-prone cells, they make attractive therapeutic targets. Targeting stem cells, or stem cell–like cells, would target the cell of origin of the cancer and have the additional advantage of enabling treatment to be based on phenotype rather than genotype. In other words, cancers would be treated on the basis of a shared property that is characteristic of all mammary stem cells. Of course, to avoid side effects, this property would need to be absent from other stem cells.
In designing specific regimens for cancer stem cells, several strategies are considered:
1) Anti–stem cell therapy: Treatment of postmenopausal women, or younger women from an at-risk group with an anti–breast stem cell therapy might severely deplete or even eliminate the breast cancer–prone cell population, with the disadvantage of mammary gland atrophy.15
2) RNA interferance: Methods of disrupting the target genes of interest within cancer models which might cause stem cell death or promote terminal differentiation can be tested in various mammary cancers.15
3) Targeting pathways that transform normal MESCs into BrCa SCs, such as the TGF-β pathway.12
4) Interferance with cancer stem cell–specific survival pathways: For example, strategies that inhibit survival mechanisms or the oxidative state of the cell may be selectively cytotoxic to BrCa Scs.23
5) Antibody-based or ligand-based therapy also appears to be a promising way to destroy cancer stem cells. A small number of target antigens on cancer stem cells have been described, and with further characterization of purified populations, additional targets are likely to become available. It remains to be determined, however, whether these and other targets will distinguish cancer stem cells from normal tissues.15

Eradication of Breast Cancer Stem Cells vs Success of Therapy
First, we must understand how therapies that effectively target the bulk of tumour cells fail to eradicate cancer stem cells. The reasons for this phenomenon may provide important clues for developing more effective and comprehensive regimens to attack both the tumour stem cells and the bulk of the disease.9,23
The recognized property of stem cells that make cancer stem cells particularly difficult to kill is that BrCa SCs reside in a largely quiescent state with regard to cell-cycle activity. Consequently, typical cytotoxic regimens that target rapidly dividing cells are unlikely to eradicate such cells.23
Selective targeting will therefore require regimens that kill cells independently of the cell cycle, or that selectively induce cycling of cancer stem cells. Another common feature of stem cells is the expression of proteins associated with the efflux of xenobiotic toxins (eg, multidrug-resistant proteins and related members of the ATP-binding cassette [ABC] transporter family). A variety of cancer cells, particularly during relapse, express such proteins, thus providing resistance to many chemotherapeutic agents.22,23
A further concern is that normal stem cells and progenitor cells may prove to be more sensitive than cancer stem cells to the effects of chemotherapy. It is critical to understand how cancer stem cells differ from normal stem cells, particularly with regard to mechanisms controlling cell survival and responses to injury. Ideally, a therapy should target pathways uniquely used by cancer stem cells to resist insults or to maintain steady-state viability.23
If a clinical remission is achieved, the presence of residual drug-resistant cancer stem cells can initiate a relapse. Hence, we must develop better methods for detection and quantitation of cancer stem cells in patients receiving cancer therapy.9
As shown in the haematopoietic system, leukaemic stem cells may be heterogeneous with respect to their self-renewal potential and quiescent state. This is also likely to be true in solid tissue cancers as well. Understanding the behaviour of cancer stem cells should better enable the design of therapies targeted at the short-lived as well as long-lived cancer stem cells. As demonstrated in breast cancer, gene profiling is a powerful tool in identifying different types of breast cancer with respect to response to therapy, relapse and metastatic potential. However, it may be necessary to profile the tumour stem cells from these different types of breast cancer as well in order to determine more appropriate therapeutic approaches.23
Most likely there will not be a single magic bullet. Once unique pathways are identified, combination therapy may be more effective than single therapy, based on the observation that cancer stem cells may be heterogeneous with respect to their quiescent state and proliferation capacity as well as the mechanisms underlying their transformation. Therefore, the future of cancer treatment may require individualized combination therapies targeting various unique pathways that are active in cancer stem cells.
Conclusion
• Cell surface markers to identify putative breast cancer stem cells are the CD44+/CD24− subset in breast cancer tissue.
• The origins of putative breast cancer stem cells are mammary epithelial stem cells and transit amplifying cells.
• Factors transforming MESCs into putative BrCa SCs are aberrent signalling pathways: TGF-β, Wnt, Notch and Hh. Another factor is the interaction with mammary stroma.
• Possible mechanisms of eradication are anti–stem cell therapy, RNA interference, interfering with signalling pathways and immunotherapy (antibody ligand–based therapy).
• Successful therapy considers eradication of BrCa SCs as a significant factor but not the only factor in treating the patients.

Acknowledgement
The author wishes to thank Professor Jeanne Adiwinata Pawitan, PhD, from the Department of Histology, Faculty of Medicine, University of Indonesia, for her guidance in writing this article, for providing valuable insight in the comprehension of the cytogenetic basis of breast cancer and cancer stem cells, and for giving constructive advice on scientific writing.

About the Author
Dr Antarianto, is a junior academic staff at the Department of Histology, Faculty of Medicine, University of Indonesia, Indonesia.
E-mail: dr.radiana.antarianto@gmail.com


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True/False Questions
1. The lifetime chance of a woman developing breast cancer is about 12%. (T)
2. Asian women have a higher rate of breast cancer than Caucasians. (F)
3. The acute myelogenous leukaemia cell was the first tumourigenic cell identified. (T)
4. Stem cells have the potential for self-renewal, developing into multiple lineages and extensive proliferation. (T)
5. Human breast cancer stem cells have been found to produce breast cancer when transplanted into mice. (T)
6. The CD44+CD24− subset of cancer cells is a homogeneous population of cancer cells. (F)
7. Putative breast cancer stem cells originate from mammary epithelial stem cells and transit amplifying cells. (T)
8. Typical cytotoxic regimens that target rapidly dividing cells are likely to eradicate breast cancer stem cells. (F)
9. Gene profiling is useful in identifying the metastatic potential of a particular type of breast cancer. (T)
10. Aberrant signalling pathways of TGF-β, Wnt, Notch and Hedgehog can transform mammary epithelial stem cells into putative breast cancer stem cells. (T)