ANZ J. Surg. 2007; 77: 464–468
doi: 10.1111/j.1445-2197.2007.04096.x
SCIENCE FOR SURGEONS
CANCER STEM CELLS: A REVIEW JOHN B. SPILLANE
AND
MICHAEL A. HENDERSON
Department of Surgical Oncology, Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia Research has been increasing in recent years into the application of stem cell biology to clinical medicine, particularly its role in the evolution and metastasis of tumours. Stem cells may be the target cell for malignant transformation, and tumour formation could be considered a disorder of stem cell self-renewal pathways. Cancer stem cells have been identified in acute myeloid leukaemia and in breast and central nervous system tumours. Cancer stem cells may have a specific role in tumour metastasis, and their understanding may provide insights into the development of predictive and prognostic markers and specific therapeutic interventions. Key words: acute myeloid leukaemia, breast cancer, cancer stem cells, central nervous system tumours, stem cells; ER, oestrogen receptor.
Abbreviations: AML, acute myeloid leukaemia; CNS, central nervous system; NOD/SCID mice, nonobese diabetic severe combined immunodeficiency disease mice.
INTRODUCTION There has been a growing interest in recent years in the role stem cells play in health and disease. With a greater understanding of their biology, a major role for stem cells in the malignant process has been proposed. Cancer stem cells have been implicated in the initiation and development of malignancy including metastasis and are the subject of this review. They may explain why standard oncology treatments sometimes fail and possibly provide an insight into designing new targeted therapies for malignant disease.1
STEM CELLS AND CANCER STEM CELLS A stem cell is defined by two important characteristics. First, it undergoes self-renewal division (Fig. 1). This is an asymmetric division in which an exact copy is produced with developmental potential identical to that of the cancer stem cell. The second characteristic is the production of daughter or ‘progenitor’ cells. They initially retain many of the characteristics of their parent stem cell but sequentially lose their self-renewing potential with each subsequent cell division as they differentiate to generate mature cells of at least one but often many cell types within an organ.2–4 This contrasts with pluripotent stem cells, which are derived from embryos and can in theory give rise to every cell type within the body.3 In most adult tissues, stem cells are rare and therefore difficult to study.2 They also differentiate into fewer cell types compared with the pluripotent stem cells.4 Studies in human and rat mammary glands suggest that stem cells are located in specific locations or stem cell niches.5 J. B. Spillane MB BS, FRACS; M. A. Henderson MB BS, MD, FRACS. Correspondence: Dr John B. Spillane, Department of Surgical Oncology, Peter MacCallum Cancer Centre, St Andrew’s Place, East Melbourne, Vic. 3002, Australia. Email:
[email protected] Accepted for publication 17 December 2006. Ó 2007 Royal Australasian College of Surgeons
A unique microenvironment is found in the stem cell niche which is required for stem cell function. Isolating stem cells from the stem cell niche may alter their function, and this needs to be considered when studying stem cells in in vitro and in vivo situations.5 Stem cells also play an important role in tissue repair and homeostasis. They potentially offer an unlimited source of specific cells for treating a variety of diseases and disabilities.1,3 Table 1 summarizes the major differences between somatic or tissue stem cells and cancer stem cells. Somatic stem cells generate normal tissues in a highly regulated process, whereas cancer stem cells produce tumours.1,2,4,6,7 The traditional model of cancer development suggests that tumours arise from a series of sequential mutations resulting from genetic instability and/or environmental factors affecting normal cells (Fig. 2). Tumour cells progress through a preneoplastic into a neoplastic phase and subsequently metastasize.4,8 Previous research has focussed on understanding the genetic changes directing a cell towards a malignancy. Although this has lead to a greater understanding of tumour behaviour, there has been less research into which cells are affected by these mutations.2,4 Recent evidence suggests that cancer stem cells or their immediate progenitor cells may be sites for this initial mutation. A major hurdle for the traditional model of cancer development is the prolonged period required to develop the first mutation that subsequently leads to malignant tumour formation. In many tissues in which tumours arise (e.g. gastrointestinal tract, epithelium, skin, blood), mature cells have a short lifespan and a limited opportunity to accumulate the multiple mutations required for tumour development.1,2 Consequently, the probability of an individual cell accumulating the necessary mutations is small.2,8 An alternative explanation that has recently been proposed is the stem cell model of tumour formation. It proposes that long-lived tissue stem cells undergo mutations that deregulate normal self-renewal pathways, leading to tumour formation.4 Although cancer stem cells are only a small subset of the total number of cells within a tumour, they have the capacity to proliferate extensively.2,4,8 In this hypothesis,
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Fig. 1. Stem cells create an exact copy of themselves and an EP cell when they divide. The EP cell then progresses to a late progenitor cell and then to the definitive cell line. DTL, definitive tissue line; EP, early progenitor; LP, late progenitor; SC, stem cell. Table 1.
Comparison of somatic and cancer stem cells
Somatic stem cell Self-renew, highly regulated Differentiate, produces mature tissue Migrate to distant tissues Long lifespan Resistant to apoptosis
Cancer stem cell Self-renew, poorly regulated Differentiate, produces tumour Metastasize to distant sites Long lifespan Resistant to apoptosis
malignant tumours are considered a disease of deregulated self-renewal.2 Studies on leukaemia and multiple myeloma indicate that a very small fraction of cells within these tumours can proliferate extensively.2 These results suggest that malignant tumours consist of a very small group of cells with enormous proliferative potential and a much larger pool of differentiated tumour cells with a limited proliferative potential.2 Cancer stem cells were first described in patients with acute myeloid leukaemia (AML). Malignant tumours comprise of a highly heterogenous population of cells that can be separated on the basis of cell surface marker antigen expression by flow cytometry.6–8 Studies have shown that a subset of human AML cells with CD34+CD382 surface antigens, which made up approx-
Fig. 2. (a) The traditional model of tumour formation. A series of mutations affect a mature cell, causing it to become malignant. Any cell has the potential to form a tumour. (b) Mutation only at the stem cell or progenitor cell level. The cancer stem cell replicates forming an exact copy of itself as well as a continuous supply of heterogenous tumour cells. DTL, mature definitive tissue cell; SC, stem cell; T, tumour.
imately 0.2% of the total AML cell population, could transfer leukaemia when transplanted into nonobese diabetic severe combined immunodeficiency disease (NOD/SCID) mice.9 Other AML cells were unable to induce leukaemia even when transplanted in larger numbers.2,9–11 The AML CD34+CD382 cell surface marker phenotype was similar to the normal haematopoietic stem cell surface marker, suggesting that AML arose from haematopoietic stem cells.2,9,10,12 The CD34+CD382 AML cells, when isolated from the general population of AML cells, gave rise to a heterogeneous leukaemic tumour mass where most cells lacked the ability to proliferate extensively except the AML stem cells.2,9 They could be serially transplanted into mice with the development of, on all occasions, a leukaemia with morphology and cell surface phenotype identical to that of the original tumour from which the stem cell arose.9 Cancer stem cells have subsequently been identified in breast and central nervous system (CNS) cancers using similar experiments.2,8 (Fig. 3). In breast cancer, cells expressing the CD44+CD24-lineage negative marker consistently formed tumours when injected into NOD/SCID mice.2,7 This subgroup made up only a small percentage of the total breast cancer mass. As few as 200 CD44+CD24-lineage negative cancer cells could consistently form breast cancers in mice compared with the failure of tumour formation following the injection of thousands of cancer cells with other cell surface markers.2,7 The CD44+CD24lineage negative cells could be injected into further mice, again with the formation of a tumour that strongly resembled the primary breast cancer on each occasion.2,7 On the basis of this work, cells having the CD44+CD24-lineage negative phenotype were confirmed as cancer stem cells in a breast cancer model.7 Similarly, both in vitro and in vivo experiments with human glioblastoma and medulloblastomas have shown that the subset of cells expressing the human neural stem cell marker CD133 accounted for almost all the proliferative activity of the tumour.2,13–16 As few as 100 CD133+ cells could be serially transplanted in NOD/SCID mouse brains, forming a tumour with the same phenotype as the original tumour, compared with the injection of 100 000 CD1332 cells that could not form CNS tumours.16 These data support the hypothesis for a cancer stem cell origin for CNS tumours.2,16,17
STEM CELLS PATHWAYS Tissue stem cells use multiple signalling pathways to control normal stem cell self-renewal. Deregulation of these pathways
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Fig. 3. Diagram outlining the experiment proving that CD44+CD24-lineage negative cells are cancer stem cells in breast cancer. (a) Using flow cytometry techniques, breast cancers are divided into subgroups of similar cells according to their cell surface markers. Each subgroup was tested by the same model. CD44+CD24-lineage negative tumour cells are separated from the main tumour mass. (b) CD44+CD24-lineage negative cells are injected into the mammary fat pad in an immune compromised (nonobese diabetic severe combined immunodeficiency disease mice) mouse. (c) Breast cancers developed in the mammary gland at the site of injection. These tumours are identical to the host breast cancer that was initially used to isolate the CD44+CD24-lineage negative cells. These cells made up only a small portion of the total breast cancer mass. (d) CD44+CD24-lineage negative cells were isolated from the mammary gland cancer of mouse shown in (c). (e) When injected into the mammary fat pad of another immunocompromised mouse, again a breast cancer developed. It was identical to both the host breast cancer and the cancer in shown in (c). This experiment can be repeated multiple times with an identical breast cancer developing on each occasion. The CD44+CD24-lineage negative cells were identified to a cancer stem cell.
may lead to neoplastic proliferation with the development of a cancer stem cell.5,6 Numerous signalling pathways have been implicated in this process including Notch, Wnt, LIF (leukaemia inhibitory factor), PTEN (phosphatase and tensin homologue deleted from chromosome 10), SHH (sonic hedgehog) and BMI1.2,4,6,7,18 The Notch pathway is important in haematopoietic and mammary epithelial stem cells and is implicated in lymphoblastic leukaemia and breast cancer.6 The Wnt pathway is implicated in colorectal cancer, lymphoblastic leukaemia, pilomatricoma, medulloblastoma and in prostate, ovarian and breast tumours, as well as in maintaining the integrity of the stem cell niche.1,5,7,19–21 Molecules in the Wnt pathway have both prooncogenic and tumoursuppressor roles.7 Overexpression of Wnt in mouse mammary gland models produces an increase in breast cancer formation.7,19 Further study into these and other mechanisms controlling selfrenewal pathways is needed to understand not only what drives tumour formation from cancer stem cells but also what mechanisms could be used to ‘switch off’ tumour formation.
STEM CELLS AND HETEROGENEITY Tumours are heterogeneous, but the mechanisms underlying this are unclear.1 Heterogeneity may result from mutations occurring early or late in a stem cell’s maturation. For example, chronic
myeloid leukaemia is believed to derive from an early stem cell progenitor because its cytogenic marker (BCR-ABL) is present in several cell lineages, for example lymphoid, myeloid and platelet cells. However, acute promyelocytic leukaemia may result from an abnormality in a late stem cell progenitor in the myeloid lineage at the promyelocytic stage.1 Tumours derived from an early stem cell may develop a more heterogenous phenotype and have an increased metastatic potential.1 Mutations in late progenitor stem cells may lead to tumours of a single cell type with reduced metastatic potential (Fig. 4).1 As recently shown in an experiment by Shackleton et al., the mammary gland develops by differentiation from its mammary stem cell.19 A diverse range of breast cancers may, therefore, develop depending on where a mutation occurs in this pathway.7,22 Consequently, a stem cell model for oestrogen receptor (ER) expression in breast cancer has been proposed, dividing breast cancer into three types, in an attempt to explain how ER-positive, ER-negative or heterogenous receptor status tumours can be created by mutations in the stem cell or progenitor cell populations (Fig. 5).7,22 In early fetal life, stem cells are ER negative, but presumably under the influence of environmental factors including oestrogen, progenitor cells that are both ER positive and ER negative can be identified at various times during growth, in particular during puberty and pregnancy.22 Ó 2007 Royal Australasian College of Surgeons
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of the tumour will differentiate into ER-positive cells.22 Antioestrogen therapy can produce a decrease in tumour size; however, the effect is short lived as ER-negative stem cells are unaffected and tumour proliferation continues despite hormonal therapy.22 This may explain why some ER-positive breast cancers continue to grow despite adjuvant hormonal therapy.22 Type 3 tumours are well differentiated and result from mutations in ER-positive progenitor cells. Hormone replacement therapy use increases the risk of cancer formation.22 They respond best to antioestrogen therapy and have the best prognosis.22
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Fig. 4. In the stem cell model, only the stem cells or their progenitor cells have the ability to form tumours. Tumour characteristics vary depending on which cell undergoes the malignant transformation. DTL, definitive tissue line; EP, early progenitor; LP, late progenitor; SC, stem cell.
Type 1 tumours develop from mutations in ER-negative stem/ progenitor cells, blocking differentiation and preventing the development of ER-positive progenitors.22 These tumours are poorly differentiated and appear to be more aggressive with a poorer prognosis. Less than 10% of these tumours are ER positive.22 Type 2 tumours are also derived from mutations in the ERnegative stem/progenitor cells. However, a variable percentage
The identification of cancer stem cells has potential therapeutic implications.7 As stem cells are important for tissue growth and repair, they have developed highly efficient mechanisms for resistance to apoptosis4 Many have overexpression of antiapoptotic genes such as Bcl-2 and may express drug-resistance transporter proteins such as MDR1 and ABC transporters.4,6,23,24 These mechanisms permit normal stem cells to become resistant to chemotherapy.17,25–27 It has been proposed that cancer stem cells also express these proteins at higher levels than the bulk population of tumour cells and may be more resistant to chemotherapeutic agents, permitting the reproliferation of tumours after chemotherapy.2,4 Developing targeted therapies that are selectively toxic to cancer stem cells while sparing normal stem cells may lead to more effective treatment options for eradicating this crucial population of cells.6 There has been much interest in microarray analysis of tumours, allowing tumour subtyping, the development of prognostic and predictive markers and the possibility of developing specific tumour treatments.28–30 The identification of stem cells in AML, breast cancers and CNS tumours raises the possibility that
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Fig. 5. Three proposed models explaining how the point at which a mutation occurs will alter the oestrogen receptor status of a breast cancer. DTL, definitive tissue line; ER, oestrogen receptor; SC, stem cell. Ó 2007 Royal Australasian College of Surgeons
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decision-making on the basis of microarray analysis of the bulk tumour population may not be entirely appropriate because the gene expression profile of the cancer stem cell may be different to the rest of the tumours.2,6 By comparing gene expression profiles of cancer stem cells, the bulk tumour cell population, normal stem cells and normal tissue, it may be possible to identify therapeutic targets that preferentially attack cancer stem cells.2 Understanding cancer stem cell biology may lead to insights into the causes and treatment of tumour metastasis. The metastatic ability of a tumour cell may be related to properties of the stem cell of origin.4 For example, the cytokine receptor CXCR4 is expressed on haematopoietic stem cells and interacts with cytokines CXCL12/SCDF that are secreted by bone marrow stromal cells. This attracts haematopoietic stem cells to the bone marrow.4,7 The CXCR4 cytokine is also overexpressed on metastatic breast cancer cells. This may direct them to the bone marrow and may be one of several potential explanations for the increased incidence of bone metastases in breast cancer.4
CONCLUSION Recent research suggests that tumour formation may result from the development of cancer stem cells by the deregulation of normal self-renewal pathways of tissue stem cells. The discovery of cancer stem cells in AML, breast cancer and some CNS tumours offers a new approach to understanding the biology of these conditions. Further study is needed to understand both normal and cancer stem cell development and whether cancer stem cells are present in other tumour types. Ultimately, new prognostic and predictive markers, as well as targeted therapeutic strategies, may be developed to force tumours into permanent remission.
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