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Normal stem cells and cancer stem cells: similar and different
Seminars in Cancer Biology 20 (2010) 85–92
Contents lists available at ScienceDirect
Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer
Review
Normal stem cells and cancer stem cells: similar and different Mark Shackleton a,b,∗ a b
Melanoma Research Laboratory and Department of Hematology and Medical Oncology, Peter MacCallum Cancer Centre, East Melbourne 3002, Australia Department of Pathology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville 3010, Australia
a r t i c l e
i n f o
Keywords: Stem cell Cancer stem cell Cancer progression Self-renewal Plasticity Interconversion Cancer regression
a b s t r a c t The functional capabilities of normal stem cells and tumorigenic cancer cells are conceptually similar in that both cell types are able to proliferate extensively. Indeed, mechanisms that regulate the defining property of normal stem cells – self-renewal – also frequently mediate oncogenesis. These conceptual links are strengthened by observations in some cancers that tumorigenic cells can not only renew their malignant potential but also generate bulk populations of non-tumorigenic cells in a manner that parallels the development of differentiated progeny from normal stem cells. But cancer cells are not normal. Although tumorigenic cells and normal stem cells are similar in some ways, they are also fundamentally different in other ways. Understanding both shared and distinguishing mechanisms that regulate normal stem cell proliferation and tumor propagation is likely to reveal opportunities for improving the treatment of patients with cancer. © 2010 Elsevier Ltd. All rights reserved.
1. Normal and cancer stem cells The fields of stem cell biology and cancer biology share a common interest: how do cells proliferate? In stem cell biology, a major focus is on a specific type of cell proliferation called self-renewal that is characteristic of stem cells. Self-renewal enables stem cells to produce at least one progeny with a similar developmental potential [1,2]. Much has been learned about the mechanisms that regulate self-renewal of normal tissue stem cells. One consistent observation in these studies has been the striking association between deregulation of stem cell function and carcinogenesis; many genes that promote self-renewal are also oncogenes and many genes that inhibit self-renewal are also tumor suppressor genes. These observations have led to the idea that some cancers originate in cells that have intrinsic self-renewal activity (i.e. stem cells) or in non-stem cells in which self-renewal is activated by oncogenic mechanisms. Studying normal self-renewal is thus important for understanding oncogenesis. Distinct from the idea that cancers originate through activation of self-renewal mechanisms is the cancer stem cell (CSC) model of malignant propagation. The CSC model refers not to the cellular origin of cancers, but to the means through which established
Abbreviations: CSC, cancer stem cell; HF, hair follicle; HSC, hematopoietic stem cell; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; Hh, Hedgehog. ∗ Correspondence address: Melanoma Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne 3002, Australia. Tel.: +61 3 9656 5235; fax: +61 3 9656 1411. E-mail address: [email protected]. 1044-579X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2010.04.002
cancers propagate themselves [3,4]. In the CSC model, cancers are composed of functionally distinct cells: those with the potential for tumor formation and those that derive from tumorigenic cells and may even retain some proliferative activity, but have lost the potential to form tumors. Extensive evidence, presented in numerous previous reviews [1,3,5], indicates that at least some cancers follow a CSC model and contain tumorigenic cells as well as nontumorigenic cells that derive from them. Central to the CSC model is the idea that non-tumorigenic cells in a cancer derive from parent tumorigenic cells in a hierarchical and stable manner that parallels in concept the development of differentiated cells from stem cells in normal tissue development and homeostasis [6]. Indeed, the CSC model is so named because of this conceptual parallel. However, it is important to limit the conceptual links between normal tissue stem cells and tumorigenic cells in the CSC model, as these cells are fundamentally different in several important ways. Normal stem cells are notable for the vigilance with which their proliferation is controlled and for the care with which their genomic integrity in maintained. Tumorigenic cells are frequently distinguished by their lack of control of such processes. Identifying differences between normal stem cells and tumorigenic cancer cells is important for understanding how cancers progress and for translating advances in CSC biology into therapies that help patients. 2. Models of cancer propagation Before considering similarities and differences between normal stem cells and tumorigenic cancer cells, it is worthwhile reviewing key concepts in the field of cancer propagation. Different models
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Fig. 1. Models of cancer propagation. (A) In the cancer stem cell (CSC) model, infrequent tumorigenic cells (colored red, renewal of malignant potential indicated by circular arrow) generate not only more tumorigenic cells but phenotypically distinct (non-red cells) non-tumorigenic cells in a manner which is stable and hierarchical (indicated by double-head arrows). (B) In the clonal evolution model, many phenotypically distinct cancer cells have malignant potential and some cells gain an advantage in disease-propagating ability by acquiring additional genetic mutations (indicated by jagged arrows). (C) In the interconversion model, although many cells have intrinsic malignant potential, cells can interconvert (indicated by two-way arrows) between actively malignant and relatively quiescent states which may be associated with phenotypic differences between cells. The large, central, two-way arrows depict the notion that these models are not mutually exclusive. Tumorigenic cells in the CSC model may undergo further genetic changes and/or may interconvert between more and less actively malignant states. Similarly, tumorigenic cells undergoing clonal evolution may transiently alter their malignant behaviour by interconversion. These changes may or may not result in a change in the predominant model of propagation that is used by a cancer. There is evidence that some cancers propagate predominantly according to a CSC model. Other cancers containing high proportions of cells with tumorigenic potential are likely to propagate predominantly by clonal evolution and/or interconversion.
are proposed to explain how established cancers propagate themselves. Classically, these include the CSC model, the clonal evolution model (also called the stochastic model) and the interconversion model (Fig. 1). Each of these models explains anecdotal and experimental observations that many cancer cells in tumors – just like cells in normal organs – are phenotypically and functionally heterogeneous. The CSC model, outlined above, is fundamentally different from the other models in that it accounts for the possibility of irreversible loss of tumorigenic potential in some cancer cells through hierarchically determined mechanisms. In practice, tumorigenic cells in the CSC model should also be relatively rare in a cancer, as when these cells comprise a high proportion of cells there is little to be gained in considering them separately from the whole cancer. Furthermore, tumorigenic cells should be distinguishable from non-tumorigenic cancer cells in a way that makes their separation for independent evaluation possible. If tumorigenic and non-tumorigenic cells were unable to be separated, the CSC model would be neither useful nor testable. It is important in the study of any cancer to consider the possibility that it may follow a CSC model, as the identification, separation and study of rare populations of tumorigenic cells distinct from bulk populations of irreversibly non-tumorigenic cells will facilitate understanding of the mechanisms of progression of these cancers. The clonal evolution model of cancer propagation [7–10] is based on the genetic instability of cancer cells. In the clonal evolution model, a high proportion of cancer cells has tumorigenic potential and individual cells gain an advantage in malignant behaviour over
other cancer cells by acquiring additional genetic mutations (Fig. 1). Intratumoral genetic heterogeneity has been observed in several cancers [11–17]. This indicates that genetically divergent clones can arise from tumorigenic cells within a cancer and independently maintain malignant potential, although the degree and rate at which tumorigenic cells undergo genetic change is unknown and may be different for different cancers at different stages of disease progression. Clonal evolution also provides a basis for understanding genetic mechanisms of therapy resistance that can be acquired by cancer cells [18–21]. The interconversion model addresses the ability of tumorigenic cancer cells to interconvert between less and more actively malignant/proliferative states. Although direct evidence (i.e. the in vivo observation of malignant cells switching between different phenotypic and malignant fates in the same tumor) is lacking, intra-vital studies showing associations between Brn-2 expression, pigmentation and motility in melanoma cells within tumors strongly suggest that interconversion occurs [22]. Interconversion was recently proposed to explain differences between tumorigenic and non-tumorigenic cells in the CSC model, raising the notion that tumorigenicity can be contextual [23]. In fact, the possibility of twoway interconversion between tumorigenic and non-tumorigenic cells was never considered in the CSC model because this model only ever addressed the intrinsic potential of cancer cells to form tumors. A cell that is non-tumorigenic in one context but becomes tumorigenic in another context has by definition not lost tumorigenic potential. Such a cell is considered tumorigenic in the CSC model, as its lack of tumorigenicity at particular point in time is reversible, context dependent and not absolute.
3. Unifying model of cancer propagation In the broad consideration of cancer propagation, the cancer stem cell model, the clonal evolution model and the interconversion model should not be thought of as mutually exclusive (Fig. 1). Cancers that follow a CSC model contain rare tumorigenic cells that may undergo clonal evolution and/or may interconvert between different states of malignant behaviour and therapy resistance. Examples of these cancers are myelogenous leukemias, in which there is evidence for hierarchical organization of tumorigenic and non-tumorigenic cells [24–28], and evidence that the tumorigenic cells are subject to ongoing genetic changes and epigenetic alterations [18,19,29] that help propagate the disease. On the other hand, cancers with high proportions of tumorigenic cells may propagate primarily by clonal evolution and/or may contain cells that use reversible mechanisms to interconvert between more and less actively malignant fates. For example, human metastatic melanomas [30] and some mouse hematopoietic malignancies [31,32] contain high proportions of tumorigenic cells. These diseases thus do not appear to follow a CSC model characterized by rare tumorigenic cells. In the case of melanoma, extensive phenotypic heterogeneity may exist among cells in a tumor, despite the cells being similarly tumorigenic [30]. The basis of this heterogeneity is unknown, but likely to be in part determined by reversible epigenetic mechanisms. As above, melanoma cells may interconvert between various phenotypic and functional fates within tumors [22]. Additionally, genetic differences between melanoma cells could also explain the phenotypic heterogeneity of this disease, although genetic variation among melanoma cells has only been evaluated in a small number of genomic regions [17] and the full extent of genetic heterogeneity in melanoma, and indeed among cells in other cancers, remains unknown. The identification of genetic and epigenetic determinants of cancer cell phenotypes and cancer cell propagation is relevant and important regardless of the mode of disease propagation.
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4. Genomic integrity in normal stem cells and tumorigenic cells The maintenance of genomic integrity in normal stem cells is critical to their function and to the well being of the organism [2]. If normal stem cells could easily acquire genetic mutations then the consequences would be dire, as these mutations would be continually passed to multiple progeny, potentially disrupting normal organ function and imparting a long-term risk of malignant transformation. For example, the delayed but high incidence of cancers in individuals exposed to high levels of radiation [33] may be attributable to genetic mutations acquired in long-lived tissue-specific stem cells at the time of radiation exposure. Normal stem cells thus have carefully regulated mechanisms to maintain genomic integrity [2,34]. In contrast, tumorigenic cells may be very different beasts. The ability to acquire and retain additional genetic mutations is present in at least some tumorigenic cancer cells. In fact, this ability is likely to be a determinant of their evolutionary success in some patients by providing the ability to produce malignant clones that can adapt to changing environmental circumstances (e.g. metabolic stress, anti-tumor immune effects, anti-neoplastic therapy) and an inexorable tendency to increasingly malignant behaviour. Although clonal evolution has been considered as an alternative to the CSC model of malignant propagation, in fact clonal evolution likely occurs to some degree in all tumorigenic cancer cells, regardless of the model of disease propagation. In cancers that contain high proportions of tumorigenic cells and do not follow a CSC model, clonal evolution is likely to be the dominant means by which these cancers propagate in patients. However, even in some cancers that do follow a CSC model in which rare tumorigenic cells gives rise to bulk populations of cells with diminished tumorigenic potential, clonal evolution is likely to be important. The lack of genomic integrity in at least some tumorigenic cancer cells makes these cells fundamentally different from normal tissue stem cells.
5. Interconversion in normal stem cells and tumorigenic cells The ability of some normal tissue stem cells to interconvert between different functional states is important for their roles in adaptive homeostasis. Cell labeling studies have suggested that highly purified mouse hematopoietic stem cells (HSCs) may interconvert between dormant and activated states in conditions of hematopoietic stress and recovery there from [35–37]. Notably, CD34 expression increases in murine HSCs during conversion to an activated state, and reduces after resumption of steady state maintenance, indicating that functional interconversion of stem cells may be associated with phenotypic interconversion [38]. In the skin, different populations of epidermal stem cells display different degrees of multi-lineage differentiation depending on the requirements of the organ. For example, in steady state maintenance of skin integrity, the interfollicular epidermis is maintained predominantly by interfollicular epidermal stem cells [39] and the hair follicle (HF) is maintained predominantly by HF stem cell populations [40–42]. However, during healing of epidermal wounds, HF stem cells temporarily contribute to repair of the interfollicular epidermis [43–45] and hair follicle neogenesis in newly repaired skin can derive from interfollicular epidermal stem cells [46]. These studies indicate that normal stem cells may change their proliferative and differentiation fates in response to environmental cues. The ability of at least some normal stem cells to interconvert between different fates depending on the requirements of the organism provides great adaptability in homeostatic maintenance.
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It is likely that tumorigenic cells in at least some cancers can interconvert between different malignant fates and even different states of sensitivity to therapy (Fig. 1). For example, cell cycle studies of leukemogenic human CML cells showed heterogeneously quiescent cell populations [47]. Furthermore, AML-bearing mice administered G-CSF prior to chemotherapy showed redistribution and increased proliferation of leukemic cells within the bone marrow compartment, resulting in enhanced eradication of tumorigenic clones by chemotherapeutic agents compared to noG-CSF controls [48,49]. This suggests that some leukemogenic cells may avoid cytotoxic-induced death by adopting quiescent states that are reversible. Other studies have explored the role of microenvironmental influences in mediating conversion of tumorigenic cells between different states of proliferation and therapy resistance. Inhibition of microenvironmental homing of leukemogenic cells by blocking antibodies to adhesion molecules VLA-4 and CD44 reduced disease engraftment in transplantation models [29,50,51]. Likewise, interference with the interaction between CXCL12 on stromal cells and CXCR4 on hematopoietic cells (that is critical for normal HSC function [52,53]) by the CXCR4 antagonists AMD3465 and AMD3100 increased sensitivity of leukemia to chemotherapy [54,55]. These studies show that modulation of tumorigenic cell fate through environmental interaction may be an important means through which cancer cells proliferate and survive. Cell intrinsic mechanisms are likely to mediate conversion of some tumorigenic cancer cells to drug resistant states. Breast cancer cells induced into epithelial–mesenchymal transition by Ecadherin knockdown displayed increased resistance to paclitaxel and increased sensitivity to salinomycin, demonstrating that cancer cells may adapt morphologically and functionally distinct fates that are associated with intrinsically varied sensitivity to therapies [56]. Furthermore, reversible histone modifications that could be inhibited pharmacologically were recently shown to confer therapy resistance in subpopulations of lung cancer cells, indicating an epigenetic basis of drug resistance in some cancer cells [57]. Although the drug resistant cell population in this study was highly enriched for the cell surface marker CD133+, it should be noted that phenotypic conversion in tumorigenic cells need not have functional consequences, as neither acquisition nor loss of CD133 predicted tumorigenic potential in human melanoma cells [4]. Interconversion among tumorigenic cancer cells is an important mechanism that explains how these cells may become phenotypically heterogeneous, resist therapies and propagate in patients.
6. Cellular differentiation in normal stem cells and tumorigenic cells Self-renewal is in effect cell proliferation without loss of differentiation potential. In normal cell development, the acquisition of a differentiated phenotype is generally associated with loss of self-renewal activity and overall reduction of a cell’s proliferative potential, although some cells with differentiated features can selfrenew (e.g. lymphocytes). The least differentiated stem cells with the highest self-renewal potential are derived from the inner cell mass during early embryogenesis as embryonic stem (ES) cells. ES cells are pluripotent, possessing the capacity to differentiate into all cell types that comprise the mature organism, including tissuespecific stem cells. Tissue-specific stem cells give rise to a limited range of differentiated cell types that are generally limited to one type of tissue. They also have a more restricted – although still extensive – self-renewal potential than ES cells. A consistent observation in the normal development of many tissues is the inverse relationship between a cell’s proliferative capacity and its acquisition of features of differentiation.
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While tumorigenic cells in a cancer must derive originally from that cancer’s cell of origin, they need not resemble phenotypically that cell. Nor must tumorigenic cells resemble the normal stem cells of the organ in which the cancer arose. In fact, there is no requirement in the CSC model that tumorigenic cells be morphologically undifferentiated; features of cellular differentiation need not distinguish tumorigenic cells from non-tumorigenic cells. For example, breast cancer CSCs may resemble morphologically their non-tumorigenic derivatives [58] and non-leukemogenic cells in human AML can have blastic features [25]. Although in other cancers such as germ cell cancers and CML, loss of tumorigenic potential in cells is associated with the acquisition of features of differentiation [59–61], the inverse relationship between proliferative capacity and differentiation status that is a feature of normal cell development is not requisite in cancers that follow a CSC model.
7. Marker expression in normal stem cells and tumorigenic cells Similarly, normal stem cells and tumorigenic cancer cells arising from the same tissues need not express the same markers. Developmental biology has been greatly advanced by the discovery of markers, particularly cell surface markers that distinguish phenotypically stem cells from their differentiated progeny. These discoveries have allowed stem cells to be isolated and studied separately from their progeny using cell separation techniques such as flow-cytometry, providing critical insights into regulatory mechanisms of stem cell function. Similarly, it is pivotal to the relevance of the CSC model in cancer biology that tumorigenic and non-tumorigenic cells are separable in a way that enables prospective isolation of these cells. The hunt for markers that distinguish tumorigenic from non-tumorigenic cells has thus been keenly pursued. These studies have for a number of cancers identified markers of CSCs and in many cases found that CSCs have similar marker expression profiles to the normal stem cells of the same organ, at least for the limited range of markers evaluated. For example, human mammary stem cells and tumorigenic cells in at least some breast cancers both lack CD24 expression [58,62]. Human AML stem cells and normal human HSCs are enriched in the CD34+CD38− fraction of bone marrow [24,63]. In the mouse, both epidermal stem cells and tumorigenic cells in chemically induced squamous cell carcinomas of the skin are CD34+ [64]. However, markers of tumorigenic cells need not be the same as markers of normal stem cells in the same tissues. For example, mouse leukemias induced by fusion gene products MLL-ENL [65], MLL-AF9 [66] and MOZ-TIF2 [67] contain leukemogenic cells with a phenotype more similar to differentiated hematopoietic cells than HSCs [68], and tumorigenic mammary cancer cells arising in several mouse models contain distinctly lower levels of CD29 expression than seen in normal mouse mammary stem cells [69,70]. In normal development, patterns of cell marker expression are generally deterministic, such that stem cell activity in the same organ from different hosts can usually be predicted by the presence of certain markers on cells. For normal stem cells, these marker expression patterns remain more or less stable throughout development and are more or less consistent within a species. However, it is not known whether markers that distinguish tumorigenic and non-tumorigenic cells in the CSC model are broadly applicable across a large range of cancers or even within one type of cancer. CD133 was originally identified as a marker of tumorigenic cells in brain cancers that follow a CSC model [71,72]. More recently, the same marker was used to enrich tumorigenic cells in colon cancer [73,74]. Although some studies have confirmed these results [75], others have indicated that CD133 is not a general marker of
tumorigenic cells, even within the limited spectrum of one cancer type [76–80]. The possibility that tumorigenic cell phenotypes may be plastic [4] and not necessarily related to differences in tumorigenic potential further clouds the relationship between cancer cell phenotype and function. Although it is appealing to conceive a CSC model in which stable marker expression defines functional properties of cells – just as in normal development – evaluation of many more markers in many more cancers than performed to date needs to be done before the relationships between marker expression and malignant behaviour can be adequately understood. In general, cancer cell phenotypes alone cannot currently be relied upon to identify tumorigenic cells in cancers that follow a CSC model, and in vivo tumorigenesis assays in optimized models [4] remain essential for the identification of tumorigenic cells. In fact, despite the relative stability of marker expression in many types of normal stem cells, functional assays are usually performed to evaluate these cells, as ‘stemness’ at its core is a functional property of cells. The same standards should be applied to tumorigenic cells.
8. Self-renewal in normal stem cells and tumorigenic cells Although self-renewal is frequently cited as a characteristic of tumorigenic cells in the CSC model, it has never been shown definitively that CSCs can self-renew in the strict definitional sense that has been applied to normal tissue stem cells. For normal stem cells, the gold-standard test of self-renewal requires the clonal in vivo demonstration of self-renewal and multi-lineage differentiation in primary transplants of stem cells, followed by demonstration of the same properties in serial transplants of the same cells [81]. In fact, due to technical limitations, self-renewal in normal tissues stem cells has only been evaluated this rigorously in a limited number of organs [70,82–87]. Nevertheless, evaluations of self-renewal activity by other methods in cells from these and other organs have provided important insights into the regulation of cell self-renewal mechanisms, which are frequently shared among tissue types. Although many stem cells have a remarkable capacity for self-renewal that supports the lifespan and homeostatic requirements of the host organ, tissue-specific stem cell exhaustion has been demonstrated. For example, there is a limit to the ability of hematopoietic stem cells [88,89] and mammary stem cells [90,91] to be serially transplanted. Self-renewed progeny of at least some tissues-specific stem cells are therefore not exactly the same as their parental cells. Even more limitations need to be placed on the concept of self-renewal in cancer. Self-renewal in tumorigenic cancer cells has generally been evaluated by demonstration of serial transplantability of polyclonal tumors and by demonstration of similar phenotypic heterogeneity in parental and daughter tumors. Serial transplantation of polyclonal tumors could occur without selfrenewal if the tumorigenic cells possessed a high potential for non-self-renewing proliferations. Furthermore, most phenotypic evaluations of the progeny of tumorigenic cells have been limited to just a handful of markers. Compared to normal tissue-specific stem cells, the bar for demonstrating self-renewal in CSCs has thus far been set low. These observations are more than mere semantics and it is important to consider carefully the nuances of self-renewal in the context of models of cancer propagation. According to definitions of self-renewal usually applied to normal stem cells, non-self-renewing divisions in malignant propagation may be just as important – if not more important – as self-renewing divisions by providing a greater proliferative and metastatic capacity. For example, the acquisition via clonal evolution of additional mutations in tumorigenic cell progeny may augment malignant behaviour, even though by strict criteria self-renewal did not occur. Additionally,
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Fig. 2. Therapeutic implications of differences between normal stem cells and tumorigenic cells. Shared mechanisms of self-renewal in normal stem cells and renewal of malignant potential in tumorigenic cells may not be suitable therapeutic targets for cancer treatment. Such approaches could be excessively toxic by non-selectively targeting self-renewal mechanisms in normal stem cells that are critical for organ function. More appealing is the possibility of selective targeting of mechanisms that are specific to the renewal of malignant potential in cancer cells. Understanding the similarities and differences between cell renewal mechanisms in normal stem cells and tumorigenic cancer cells is thus likely to improve the treatment of cancer.
non-self-renewed tumorigenic cells could still give rise to bulk populations of non-tumorigenic cells, such that the cancer followed a CSC model. Conversely, the proliferation of tumorigenic cells that do not generate non-tumorigenic cells and thus do not follow a CSC model may still be driven by self-renewal mechanisms. True selfrenewal of tumorigenic cells is therefore not requisite in cancers that follow a CSC model, and self-renewal could occur in tumorigenic cells that are very frequent in cancers that do not follow a CSC model. From a clinical perspective, the important feature of cancer cells that is ‘self-renewed’ in all models of disease propagation is malignant potential (Fig. 2). 9. Considerations in targeting mechanisms of cell renewal in normal stem cells and tumorigenic cells Although regulatory mechanisms of self-renewal in normal stem cells also frequently regulate oncogenesis, they can also contribute to the propagation of established cancers. The polycomb repressor complex protein Bmi-1 is required for self-renewal of normal stem cells in numerous organs [92–94] and for the propagation of Hoxa9–Meis1 induced AML in mice [92]. There is evidence that the tumor suppressor p53 promotes HSC quiescence [95,96], and restoration of p53 function in established p53 deficient mouse cancers causes tumor regression [97,98]. Intact ‘Smoothened’ function, which is essential for Hedgehog (Hh) pathway signaling, facilitates the serial transplantability of HSCs and increases leukemogenic cell numbers in a model of CML [99]. In the skin, Hh signaling is required for normal hair follicle development and homeostasis [100–103]. In a model of cutaneous basal cell carcinoma characterized by excessive Hh signaling driven by constitutive expression of Gli2, inducible inhibition of Hedgehog signaling causes stable regression of established tumors [104]. Interestingly, restoration of constitutive Hh signaling in this model allows regrowth of persistent tumorigenic cells, indicating that separate mechanisms may regulate the maintenance and propagation
of cells in established tumors. These studies of shared mechanisms that promote both normal stem cell self-renewal and renewal of malignant potential in tumorigenic cells raise a potential problem in the toxicity that may occur in therapeutic targeting of these mechanisms. For example, inhibition of self-renewal pathways that drive cancer propagation may cause undue toxicity if the same pathways are important for homeostatic function in normal stem cells. One way around this is to design therapies that target mutations present only in cancer cells. Examples of this approach in the case of p53 mutations are reviewed elsewhere [105]. Another method is to target selectively mechanisms of tumor propagation that are distinct from normal stem cell regulation (Fig. 2). Fortunately, some of these have been identified. Conditional deletion in mice of the PI-3 kinase pathway regulator Pten produces leukemia that develops according to a CSC model [106]. However, Pten deletion causes a biphasic response in normal HSCs characterized by initial expansion and subsequent deletion [106,107]. This divergence of Pten-mediated PI-3 kinase pathway function in leukemogenic cells and HSCs is therapeutically exploitable by the mTor inhibitor rapamycin, which eradicates leukemogenic cells and restores HSC function in Pten-deleted hematopoietic cells [106]. Similarly, Wnt/-catenin signaling sustains the propagation of chemically induced cutaneous squamous cell cancers but is not essential for the maintenance of normal epidermal homeostasis [64,108–111]. The identification and targeting of signaling mechanisms that are specific for tumorigenic cells should provide the opportunity for selective targeting of these cells and thereby a large therapeutic index in the treatment of malignant disease (Fig. 2). 10. Conclusions Stem cell biology has made profound contributions to understanding normal human development and the development of cancer. A greater understanding of stem cells has led to the appre-
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ciation that deregulation of stem cell function plays a central role in the genesis of some cancers. Additionally, key concepts of stem cell biology are applicable to and helpful for understanding how some established cancers propagate in patients. The phrase ‘cancer stem cell’ is simultaneously catchy and informative. However, unfortunately it also encourages the notion that tumorigenic cancer cells and normal stem cells are more similar than they really are [112]. It is important to remember that it is the malignant potential of some cancer cells that harms patients, not the degree to which these cells resemble normal stem cells. The CSC model does not require that tumorigenic cells be similar phenotypically, genetically, epigenetically or functionally to the normal stem cells of the same organ. What the CSC model does require (to be relevant to cancer research and treatment) is for tumorigenic cells to be relatively infrequent in a cancer, to be capable of generating both more tumorigenic cells as well as bulk populations of non-tumorigenic cells in a stable and hierarchical manner, and to be separable from non-tumorigenic cells. In some cancers that follow a CSC model, these criteria coincide with tumorigenic cells sharing many features of normal stem cells. In other cancers that follow a CSC model, tumorigenic cells may bear little resemblance to normal stem cells from the same tissue, revealing therapeutic opportunities. The overlapping aspects of stem cell biology and cancer cell biology have provided great impetus for a better understanding of cancer genesis and propagation and opened new windows for improved treatment of patients. Although there is much yet to be learned about how mechanisms that regulate normal stem cell function may be used by cancer cells to propagate disease, these studies must be underpinned by an appreciation of the fact that cancer cells are not normal. Rather, careful consideration of the ways in which normal stem cells and tumorigenic cancer cells are both similar and different is required to maximize the potential of this research. Conflict of interest The author declares that there are no conflicts of interest. Acknowledgements Thanks to Elsa Quintana for assistance with figures, and to Jeff Magee and Boaz Levi for helpful comments. M.S. is supported by the Peter MacCallum Cancer Centre, the Australian National Health and Medical Research Council, the Victorian Cancer Agency and the Victorian Endowment for Science, Innovation and Technology. Apologies to authors whose work could not be cited due to space limitations. References [1] Lobo NA, Shimono Y, Qian D, Clarke MF. The biology of cancer stem cells. Annu Rev Cell Dev Biol 2007;23:675–99. [2] He S, Nakada D, Morrison SJ. Mechanisms of stem cell self-renewal. Annu Rev Cell Dev Biol 2009;25:377–406. [3] Dick JE. Stem cell concepts renew cancer research. Blood 2008;112:4793–807. [4] Shackleton M, Quintana E, Fearon ER, Morrison SJ. Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell 2009;138:822–9. [5] Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB. Tumourinitiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 2009;8:806–23. [6] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105–11. [7] Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194:23–8. [8] Foulds L. The natural history of cancer. J Chronic Dis 1958;8:2–37. [9] Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759–67. [10] Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998;396:643–9.
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Contents lists available at ScienceDirect
Seminars in Cancer Biology journal homepage: www.elsevier.com/locate/semcancer
Review
Normal stem cells and cancer stem cells: similar and different Mark Shackleton a,b,∗ a b
Melanoma Research Laboratory and Department of Hematology and Medical Oncology, Peter MacCallum Cancer Centre, East Melbourne 3002, Australia Department of Pathology, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville 3010, Australia
a r t i c l e
i n f o
Keywords: Stem cell Cancer stem cell Cancer progression Self-renewal Plasticity Interconversion Cancer regression
a b s t r a c t The functional capabilities of normal stem cells and tumorigenic cancer cells are conceptually similar in that both cell types are able to proliferate extensively. Indeed, mechanisms that regulate the defining property of normal stem cells – self-renewal – also frequently mediate oncogenesis. These conceptual links are strengthened by observations in some cancers that tumorigenic cells can not only renew their malignant potential but also generate bulk populations of non-tumorigenic cells in a manner that parallels the development of differentiated progeny from normal stem cells. But cancer cells are not normal. Although tumorigenic cells and normal stem cells are similar in some ways, they are also fundamentally different in other ways. Understanding both shared and distinguishing mechanisms that regulate normal stem cell proliferation and tumor propagation is likely to reveal opportunities for improving the treatment of patients with cancer. © 2010 Elsevier Ltd. All rights reserved.
1. Normal and cancer stem cells The fields of stem cell biology and cancer biology share a common interest: how do cells proliferate? In stem cell biology, a major focus is on a specific type of cell proliferation called self-renewal that is characteristic of stem cells. Self-renewal enables stem cells to produce at least one progeny with a similar developmental potential [1,2]. Much has been learned about the mechanisms that regulate self-renewal of normal tissue stem cells. One consistent observation in these studies has been the striking association between deregulation of stem cell function and carcinogenesis; many genes that promote self-renewal are also oncogenes and many genes that inhibit self-renewal are also tumor suppressor genes. These observations have led to the idea that some cancers originate in cells that have intrinsic self-renewal activity (i.e. stem cells) or in non-stem cells in which self-renewal is activated by oncogenic mechanisms. Studying normal self-renewal is thus important for understanding oncogenesis. Distinct from the idea that cancers originate through activation of self-renewal mechanisms is the cancer stem cell (CSC) model of malignant propagation. The CSC model refers not to the cellular origin of cancers, but to the means through which established
Abbreviations: CSC, cancer stem cell; HF, hair follicle; HSC, hematopoietic stem cell; AML, acute myelogenous leukemia; CML, chronic myelogenous leukemia; Hh, Hedgehog. ∗ Correspondence address: Melanoma Research Laboratory, Peter MacCallum Cancer Centre, East Melbourne 3002, Australia. Tel.: +61 3 9656 5235; fax: +61 3 9656 1411. E-mail address: [email protected]. 1044-579X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcancer.2010.04.002
cancers propagate themselves [3,4]. In the CSC model, cancers are composed of functionally distinct cells: those with the potential for tumor formation and those that derive from tumorigenic cells and may even retain some proliferative activity, but have lost the potential to form tumors. Extensive evidence, presented in numerous previous reviews [1,3,5], indicates that at least some cancers follow a CSC model and contain tumorigenic cells as well as nontumorigenic cells that derive from them. Central to the CSC model is the idea that non-tumorigenic cells in a cancer derive from parent tumorigenic cells in a hierarchical and stable manner that parallels in concept the development of differentiated cells from stem cells in normal tissue development and homeostasis [6]. Indeed, the CSC model is so named because of this conceptual parallel. However, it is important to limit the conceptual links between normal tissue stem cells and tumorigenic cells in the CSC model, as these cells are fundamentally different in several important ways. Normal stem cells are notable for the vigilance with which their proliferation is controlled and for the care with which their genomic integrity in maintained. Tumorigenic cells are frequently distinguished by their lack of control of such processes. Identifying differences between normal stem cells and tumorigenic cancer cells is important for understanding how cancers progress and for translating advances in CSC biology into therapies that help patients. 2. Models of cancer propagation Before considering similarities and differences between normal stem cells and tumorigenic cancer cells, it is worthwhile reviewing key concepts in the field of cancer propagation. Different models
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Fig. 1. Models of cancer propagation. (A) In the cancer stem cell (CSC) model, infrequent tumorigenic cells (colored red, renewal of malignant potential indicated by circular arrow) generate not only more tumorigenic cells but phenotypically distinct (non-red cells) non-tumorigenic cells in a manner which is stable and hierarchical (indicated by double-head arrows). (B) In the clonal evolution model, many phenotypically distinct cancer cells have malignant potential and some cells gain an advantage in disease-propagating ability by acquiring additional genetic mutations (indicated by jagged arrows). (C) In the interconversion model, although many cells have intrinsic malignant potential, cells can interconvert (indicated by two-way arrows) between actively malignant and relatively quiescent states which may be associated with phenotypic differences between cells. The large, central, two-way arrows depict the notion that these models are not mutually exclusive. Tumorigenic cells in the CSC model may undergo further genetic changes and/or may interconvert between more and less actively malignant states. Similarly, tumorigenic cells undergoing clonal evolution may transiently alter their malignant behaviour by interconversion. These changes may or may not result in a change in the predominant model of propagation that is used by a cancer. There is evidence that some cancers propagate predominantly according to a CSC model. Other cancers containing high proportions of cells with tumorigenic potential are likely to propagate predominantly by clonal evolution and/or interconversion.
are proposed to explain how established cancers propagate themselves. Classically, these include the CSC model, the clonal evolution model (also called the stochastic model) and the interconversion model (Fig. 1). Each of these models explains anecdotal and experimental observations that many cancer cells in tumors – just like cells in normal organs – are phenotypically and functionally heterogeneous. The CSC model, outlined above, is fundamentally different from the other models in that it accounts for the possibility of irreversible loss of tumorigenic potential in some cancer cells through hierarchically determined mechanisms. In practice, tumorigenic cells in the CSC model should also be relatively rare in a cancer, as when these cells comprise a high proportion of cells there is little to be gained in considering them separately from the whole cancer. Furthermore, tumorigenic cells should be distinguishable from non-tumorigenic cancer cells in a way that makes their separation for independent evaluation possible. If tumorigenic and non-tumorigenic cells were unable to be separated, the CSC model would be neither useful nor testable. It is important in the study of any cancer to consider the possibility that it may follow a CSC model, as the identification, separation and study of rare populations of tumorigenic cells distinct from bulk populations of irreversibly non-tumorigenic cells will facilitate understanding of the mechanisms of progression of these cancers. The clonal evolution model of cancer propagation [7–10] is based on the genetic instability of cancer cells. In the clonal evolution model, a high proportion of cancer cells has tumorigenic potential and individual cells gain an advantage in malignant behaviour over
other cancer cells by acquiring additional genetic mutations (Fig. 1). Intratumoral genetic heterogeneity has been observed in several cancers [11–17]. This indicates that genetically divergent clones can arise from tumorigenic cells within a cancer and independently maintain malignant potential, although the degree and rate at which tumorigenic cells undergo genetic change is unknown and may be different for different cancers at different stages of disease progression. Clonal evolution also provides a basis for understanding genetic mechanisms of therapy resistance that can be acquired by cancer cells [18–21]. The interconversion model addresses the ability of tumorigenic cancer cells to interconvert between less and more actively malignant/proliferative states. Although direct evidence (i.e. the in vivo observation of malignant cells switching between different phenotypic and malignant fates in the same tumor) is lacking, intra-vital studies showing associations between Brn-2 expression, pigmentation and motility in melanoma cells within tumors strongly suggest that interconversion occurs [22]. Interconversion was recently proposed to explain differences between tumorigenic and non-tumorigenic cells in the CSC model, raising the notion that tumorigenicity can be contextual [23]. In fact, the possibility of twoway interconversion between tumorigenic and non-tumorigenic cells was never considered in the CSC model because this model only ever addressed the intrinsic potential of cancer cells to form tumors. A cell that is non-tumorigenic in one context but becomes tumorigenic in another context has by definition not lost tumorigenic potential. Such a cell is considered tumorigenic in the CSC model, as its lack of tumorigenicity at particular point in time is reversible, context dependent and not absolute.
3. Unifying model of cancer propagation In the broad consideration of cancer propagation, the cancer stem cell model, the clonal evolution model and the interconversion model should not be thought of as mutually exclusive (Fig. 1). Cancers that follow a CSC model contain rare tumorigenic cells that may undergo clonal evolution and/or may interconvert between different states of malignant behaviour and therapy resistance. Examples of these cancers are myelogenous leukemias, in which there is evidence for hierarchical organization of tumorigenic and non-tumorigenic cells [24–28], and evidence that the tumorigenic cells are subject to ongoing genetic changes and epigenetic alterations [18,19,29] that help propagate the disease. On the other hand, cancers with high proportions of tumorigenic cells may propagate primarily by clonal evolution and/or may contain cells that use reversible mechanisms to interconvert between more and less actively malignant fates. For example, human metastatic melanomas [30] and some mouse hematopoietic malignancies [31,32] contain high proportions of tumorigenic cells. These diseases thus do not appear to follow a CSC model characterized by rare tumorigenic cells. In the case of melanoma, extensive phenotypic heterogeneity may exist among cells in a tumor, despite the cells being similarly tumorigenic [30]. The basis of this heterogeneity is unknown, but likely to be in part determined by reversible epigenetic mechanisms. As above, melanoma cells may interconvert between various phenotypic and functional fates within tumors [22]. Additionally, genetic differences between melanoma cells could also explain the phenotypic heterogeneity of this disease, although genetic variation among melanoma cells has only been evaluated in a small number of genomic regions [17] and the full extent of genetic heterogeneity in melanoma, and indeed among cells in other cancers, remains unknown. The identification of genetic and epigenetic determinants of cancer cell phenotypes and cancer cell propagation is relevant and important regardless of the mode of disease propagation.
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4. Genomic integrity in normal stem cells and tumorigenic cells The maintenance of genomic integrity in normal stem cells is critical to their function and to the well being of the organism [2]. If normal stem cells could easily acquire genetic mutations then the consequences would be dire, as these mutations would be continually passed to multiple progeny, potentially disrupting normal organ function and imparting a long-term risk of malignant transformation. For example, the delayed but high incidence of cancers in individuals exposed to high levels of radiation [33] may be attributable to genetic mutations acquired in long-lived tissue-specific stem cells at the time of radiation exposure. Normal stem cells thus have carefully regulated mechanisms to maintain genomic integrity [2,34]. In contrast, tumorigenic cells may be very different beasts. The ability to acquire and retain additional genetic mutations is present in at least some tumorigenic cancer cells. In fact, this ability is likely to be a determinant of their evolutionary success in some patients by providing the ability to produce malignant clones that can adapt to changing environmental circumstances (e.g. metabolic stress, anti-tumor immune effects, anti-neoplastic therapy) and an inexorable tendency to increasingly malignant behaviour. Although clonal evolution has been considered as an alternative to the CSC model of malignant propagation, in fact clonal evolution likely occurs to some degree in all tumorigenic cancer cells, regardless of the model of disease propagation. In cancers that contain high proportions of tumorigenic cells and do not follow a CSC model, clonal evolution is likely to be the dominant means by which these cancers propagate in patients. However, even in some cancers that do follow a CSC model in which rare tumorigenic cells gives rise to bulk populations of cells with diminished tumorigenic potential, clonal evolution is likely to be important. The lack of genomic integrity in at least some tumorigenic cancer cells makes these cells fundamentally different from normal tissue stem cells.
5. Interconversion in normal stem cells and tumorigenic cells The ability of some normal tissue stem cells to interconvert between different functional states is important for their roles in adaptive homeostasis. Cell labeling studies have suggested that highly purified mouse hematopoietic stem cells (HSCs) may interconvert between dormant and activated states in conditions of hematopoietic stress and recovery there from [35–37]. Notably, CD34 expression increases in murine HSCs during conversion to an activated state, and reduces after resumption of steady state maintenance, indicating that functional interconversion of stem cells may be associated with phenotypic interconversion [38]. In the skin, different populations of epidermal stem cells display different degrees of multi-lineage differentiation depending on the requirements of the organ. For example, in steady state maintenance of skin integrity, the interfollicular epidermis is maintained predominantly by interfollicular epidermal stem cells [39] and the hair follicle (HF) is maintained predominantly by HF stem cell populations [40–42]. However, during healing of epidermal wounds, HF stem cells temporarily contribute to repair of the interfollicular epidermis [43–45] and hair follicle neogenesis in newly repaired skin can derive from interfollicular epidermal stem cells [46]. These studies indicate that normal stem cells may change their proliferative and differentiation fates in response to environmental cues. The ability of at least some normal stem cells to interconvert between different fates depending on the requirements of the organism provides great adaptability in homeostatic maintenance.
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It is likely that tumorigenic cells in at least some cancers can interconvert between different malignant fates and even different states of sensitivity to therapy (Fig. 1). For example, cell cycle studies of leukemogenic human CML cells showed heterogeneously quiescent cell populations [47]. Furthermore, AML-bearing mice administered G-CSF prior to chemotherapy showed redistribution and increased proliferation of leukemic cells within the bone marrow compartment, resulting in enhanced eradication of tumorigenic clones by chemotherapeutic agents compared to noG-CSF controls [48,49]. This suggests that some leukemogenic cells may avoid cytotoxic-induced death by adopting quiescent states that are reversible. Other studies have explored the role of microenvironmental influences in mediating conversion of tumorigenic cells between different states of proliferation and therapy resistance. Inhibition of microenvironmental homing of leukemogenic cells by blocking antibodies to adhesion molecules VLA-4 and CD44 reduced disease engraftment in transplantation models [29,50,51]. Likewise, interference with the interaction between CXCL12 on stromal cells and CXCR4 on hematopoietic cells (that is critical for normal HSC function [52,53]) by the CXCR4 antagonists AMD3465 and AMD3100 increased sensitivity of leukemia to chemotherapy [54,55]. These studies show that modulation of tumorigenic cell fate through environmental interaction may be an important means through which cancer cells proliferate and survive. Cell intrinsic mechanisms are likely to mediate conversion of some tumorigenic cancer cells to drug resistant states. Breast cancer cells induced into epithelial–mesenchymal transition by Ecadherin knockdown displayed increased resistance to paclitaxel and increased sensitivity to salinomycin, demonstrating that cancer cells may adapt morphologically and functionally distinct fates that are associated with intrinsically varied sensitivity to therapies [56]. Furthermore, reversible histone modifications that could be inhibited pharmacologically were recently shown to confer therapy resistance in subpopulations of lung cancer cells, indicating an epigenetic basis of drug resistance in some cancer cells [57]. Although the drug resistant cell population in this study was highly enriched for the cell surface marker CD133+, it should be noted that phenotypic conversion in tumorigenic cells need not have functional consequences, as neither acquisition nor loss of CD133 predicted tumorigenic potential in human melanoma cells [4]. Interconversion among tumorigenic cancer cells is an important mechanism that explains how these cells may become phenotypically heterogeneous, resist therapies and propagate in patients.
6. Cellular differentiation in normal stem cells and tumorigenic cells Self-renewal is in effect cell proliferation without loss of differentiation potential. In normal cell development, the acquisition of a differentiated phenotype is generally associated with loss of self-renewal activity and overall reduction of a cell’s proliferative potential, although some cells with differentiated features can selfrenew (e.g. lymphocytes). The least differentiated stem cells with the highest self-renewal potential are derived from the inner cell mass during early embryogenesis as embryonic stem (ES) cells. ES cells are pluripotent, possessing the capacity to differentiate into all cell types that comprise the mature organism, including tissuespecific stem cells. Tissue-specific stem cells give rise to a limited range of differentiated cell types that are generally limited to one type of tissue. They also have a more restricted – although still extensive – self-renewal potential than ES cells. A consistent observation in the normal development of many tissues is the inverse relationship between a cell’s proliferative capacity and its acquisition of features of differentiation.
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While tumorigenic cells in a cancer must derive originally from that cancer’s cell of origin, they need not resemble phenotypically that cell. Nor must tumorigenic cells resemble the normal stem cells of the organ in which the cancer arose. In fact, there is no requirement in the CSC model that tumorigenic cells be morphologically undifferentiated; features of cellular differentiation need not distinguish tumorigenic cells from non-tumorigenic cells. For example, breast cancer CSCs may resemble morphologically their non-tumorigenic derivatives [58] and non-leukemogenic cells in human AML can have blastic features [25]. Although in other cancers such as germ cell cancers and CML, loss of tumorigenic potential in cells is associated with the acquisition of features of differentiation [59–61], the inverse relationship between proliferative capacity and differentiation status that is a feature of normal cell development is not requisite in cancers that follow a CSC model.
7. Marker expression in normal stem cells and tumorigenic cells Similarly, normal stem cells and tumorigenic cancer cells arising from the same tissues need not express the same markers. Developmental biology has been greatly advanced by the discovery of markers, particularly cell surface markers that distinguish phenotypically stem cells from their differentiated progeny. These discoveries have allowed stem cells to be isolated and studied separately from their progeny using cell separation techniques such as flow-cytometry, providing critical insights into regulatory mechanisms of stem cell function. Similarly, it is pivotal to the relevance of the CSC model in cancer biology that tumorigenic and non-tumorigenic cells are separable in a way that enables prospective isolation of these cells. The hunt for markers that distinguish tumorigenic from non-tumorigenic cells has thus been keenly pursued. These studies have for a number of cancers identified markers of CSCs and in many cases found that CSCs have similar marker expression profiles to the normal stem cells of the same organ, at least for the limited range of markers evaluated. For example, human mammary stem cells and tumorigenic cells in at least some breast cancers both lack CD24 expression [58,62]. Human AML stem cells and normal human HSCs are enriched in the CD34+CD38− fraction of bone marrow [24,63]. In the mouse, both epidermal stem cells and tumorigenic cells in chemically induced squamous cell carcinomas of the skin are CD34+ [64]. However, markers of tumorigenic cells need not be the same as markers of normal stem cells in the same tissues. For example, mouse leukemias induced by fusion gene products MLL-ENL [65], MLL-AF9 [66] and MOZ-TIF2 [67] contain leukemogenic cells with a phenotype more similar to differentiated hematopoietic cells than HSCs [68], and tumorigenic mammary cancer cells arising in several mouse models contain distinctly lower levels of CD29 expression than seen in normal mouse mammary stem cells [69,70]. In normal development, patterns of cell marker expression are generally deterministic, such that stem cell activity in the same organ from different hosts can usually be predicted by the presence of certain markers on cells. For normal stem cells, these marker expression patterns remain more or less stable throughout development and are more or less consistent within a species. However, it is not known whether markers that distinguish tumorigenic and non-tumorigenic cells in the CSC model are broadly applicable across a large range of cancers or even within one type of cancer. CD133 was originally identified as a marker of tumorigenic cells in brain cancers that follow a CSC model [71,72]. More recently, the same marker was used to enrich tumorigenic cells in colon cancer [73,74]. Although some studies have confirmed these results [75], others have indicated that CD133 is not a general marker of
tumorigenic cells, even within the limited spectrum of one cancer type [76–80]. The possibility that tumorigenic cell phenotypes may be plastic [4] and not necessarily related to differences in tumorigenic potential further clouds the relationship between cancer cell phenotype and function. Although it is appealing to conceive a CSC model in which stable marker expression defines functional properties of cells – just as in normal development – evaluation of many more markers in many more cancers than performed to date needs to be done before the relationships between marker expression and malignant behaviour can be adequately understood. In general, cancer cell phenotypes alone cannot currently be relied upon to identify tumorigenic cells in cancers that follow a CSC model, and in vivo tumorigenesis assays in optimized models [4] remain essential for the identification of tumorigenic cells. In fact, despite the relative stability of marker expression in many types of normal stem cells, functional assays are usually performed to evaluate these cells, as ‘stemness’ at its core is a functional property of cells. The same standards should be applied to tumorigenic cells.
8. Self-renewal in normal stem cells and tumorigenic cells Although self-renewal is frequently cited as a characteristic of tumorigenic cells in the CSC model, it has never been shown definitively that CSCs can self-renew in the strict definitional sense that has been applied to normal tissue stem cells. For normal stem cells, the gold-standard test of self-renewal requires the clonal in vivo demonstration of self-renewal and multi-lineage differentiation in primary transplants of stem cells, followed by demonstration of the same properties in serial transplants of the same cells [81]. In fact, due to technical limitations, self-renewal in normal tissues stem cells has only been evaluated this rigorously in a limited number of organs [70,82–87]. Nevertheless, evaluations of self-renewal activity by other methods in cells from these and other organs have provided important insights into the regulation of cell self-renewal mechanisms, which are frequently shared among tissue types. Although many stem cells have a remarkable capacity for self-renewal that supports the lifespan and homeostatic requirements of the host organ, tissue-specific stem cell exhaustion has been demonstrated. For example, there is a limit to the ability of hematopoietic stem cells [88,89] and mammary stem cells [90,91] to be serially transplanted. Self-renewed progeny of at least some tissues-specific stem cells are therefore not exactly the same as their parental cells. Even more limitations need to be placed on the concept of self-renewal in cancer. Self-renewal in tumorigenic cancer cells has generally been evaluated by demonstration of serial transplantability of polyclonal tumors and by demonstration of similar phenotypic heterogeneity in parental and daughter tumors. Serial transplantation of polyclonal tumors could occur without selfrenewal if the tumorigenic cells possessed a high potential for non-self-renewing proliferations. Furthermore, most phenotypic evaluations of the progeny of tumorigenic cells have been limited to just a handful of markers. Compared to normal tissue-specific stem cells, the bar for demonstrating self-renewal in CSCs has thus far been set low. These observations are more than mere semantics and it is important to consider carefully the nuances of self-renewal in the context of models of cancer propagation. According to definitions of self-renewal usually applied to normal stem cells, non-self-renewing divisions in malignant propagation may be just as important – if not more important – as self-renewing divisions by providing a greater proliferative and metastatic capacity. For example, the acquisition via clonal evolution of additional mutations in tumorigenic cell progeny may augment malignant behaviour, even though by strict criteria self-renewal did not occur. Additionally,
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Fig. 2. Therapeutic implications of differences between normal stem cells and tumorigenic cells. Shared mechanisms of self-renewal in normal stem cells and renewal of malignant potential in tumorigenic cells may not be suitable therapeutic targets for cancer treatment. Such approaches could be excessively toxic by non-selectively targeting self-renewal mechanisms in normal stem cells that are critical for organ function. More appealing is the possibility of selective targeting of mechanisms that are specific to the renewal of malignant potential in cancer cells. Understanding the similarities and differences between cell renewal mechanisms in normal stem cells and tumorigenic cancer cells is thus likely to improve the treatment of cancer.
non-self-renewed tumorigenic cells could still give rise to bulk populations of non-tumorigenic cells, such that the cancer followed a CSC model. Conversely, the proliferation of tumorigenic cells that do not generate non-tumorigenic cells and thus do not follow a CSC model may still be driven by self-renewal mechanisms. True selfrenewal of tumorigenic cells is therefore not requisite in cancers that follow a CSC model, and self-renewal could occur in tumorigenic cells that are very frequent in cancers that do not follow a CSC model. From a clinical perspective, the important feature of cancer cells that is ‘self-renewed’ in all models of disease propagation is malignant potential (Fig. 2). 9. Considerations in targeting mechanisms of cell renewal in normal stem cells and tumorigenic cells Although regulatory mechanisms of self-renewal in normal stem cells also frequently regulate oncogenesis, they can also contribute to the propagation of established cancers. The polycomb repressor complex protein Bmi-1 is required for self-renewal of normal stem cells in numerous organs [92–94] and for the propagation of Hoxa9–Meis1 induced AML in mice [92]. There is evidence that the tumor suppressor p53 promotes HSC quiescence [95,96], and restoration of p53 function in established p53 deficient mouse cancers causes tumor regression [97,98]. Intact ‘Smoothened’ function, which is essential for Hedgehog (Hh) pathway signaling, facilitates the serial transplantability of HSCs and increases leukemogenic cell numbers in a model of CML [99]. In the skin, Hh signaling is required for normal hair follicle development and homeostasis [100–103]. In a model of cutaneous basal cell carcinoma characterized by excessive Hh signaling driven by constitutive expression of Gli2, inducible inhibition of Hedgehog signaling causes stable regression of established tumors [104]. Interestingly, restoration of constitutive Hh signaling in this model allows regrowth of persistent tumorigenic cells, indicating that separate mechanisms may regulate the maintenance and propagation
of cells in established tumors. These studies of shared mechanisms that promote both normal stem cell self-renewal and renewal of malignant potential in tumorigenic cells raise a potential problem in the toxicity that may occur in therapeutic targeting of these mechanisms. For example, inhibition of self-renewal pathways that drive cancer propagation may cause undue toxicity if the same pathways are important for homeostatic function in normal stem cells. One way around this is to design therapies that target mutations present only in cancer cells. Examples of this approach in the case of p53 mutations are reviewed elsewhere [105]. Another method is to target selectively mechanisms of tumor propagation that are distinct from normal stem cell regulation (Fig. 2). Fortunately, some of these have been identified. Conditional deletion in mice of the PI-3 kinase pathway regulator Pten produces leukemia that develops according to a CSC model [106]. However, Pten deletion causes a biphasic response in normal HSCs characterized by initial expansion and subsequent deletion [106,107]. This divergence of Pten-mediated PI-3 kinase pathway function in leukemogenic cells and HSCs is therapeutically exploitable by the mTor inhibitor rapamycin, which eradicates leukemogenic cells and restores HSC function in Pten-deleted hematopoietic cells [106]. Similarly, Wnt/-catenin signaling sustains the propagation of chemically induced cutaneous squamous cell cancers but is not essential for the maintenance of normal epidermal homeostasis [64,108–111]. The identification and targeting of signaling mechanisms that are specific for tumorigenic cells should provide the opportunity for selective targeting of these cells and thereby a large therapeutic index in the treatment of malignant disease (Fig. 2). 10. Conclusions Stem cell biology has made profound contributions to understanding normal human development and the development of cancer. A greater understanding of stem cells has led to the appre-
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ciation that deregulation of stem cell function plays a central role in the genesis of some cancers. Additionally, key concepts of stem cell biology are applicable to and helpful for understanding how some established cancers propagate in patients. The phrase ‘cancer stem cell’ is simultaneously catchy and informative. However, unfortunately it also encourages the notion that tumorigenic cancer cells and normal stem cells are more similar than they really are [112]. It is important to remember that it is the malignant potential of some cancer cells that harms patients, not the degree to which these cells resemble normal stem cells. The CSC model does not require that tumorigenic cells be similar phenotypically, genetically, epigenetically or functionally to the normal stem cells of the same organ. What the CSC model does require (to be relevant to cancer research and treatment) is for tumorigenic cells to be relatively infrequent in a cancer, to be capable of generating both more tumorigenic cells as well as bulk populations of non-tumorigenic cells in a stable and hierarchical manner, and to be separable from non-tumorigenic cells. In some cancers that follow a CSC model, these criteria coincide with tumorigenic cells sharing many features of normal stem cells. In other cancers that follow a CSC model, tumorigenic cells may bear little resemblance to normal stem cells from the same tissue, revealing therapeutic opportunities. The overlapping aspects of stem cell biology and cancer cell biology have provided great impetus for a better understanding of cancer genesis and propagation and opened new windows for improved treatment of patients. Although there is much yet to be learned about how mechanisms that regulate normal stem cell function may be used by cancer cells to propagate disease, these studies must be underpinned by an appreciation of the fact that cancer cells are not normal. Rather, careful consideration of the ways in which normal stem cells and tumorigenic cancer cells are both similar and different is required to maximize the potential of this research. Conflict of interest The author declares that there are no conflicts of interest. Acknowledgements Thanks to Elsa Quintana for assistance with figures, and to Jeff Magee and Boaz Levi for helpful comments. M.S. is supported by the Peter MacCallum Cancer Centre, the Australian National Health and Medical Research Council, the Victorian Cancer Agency and the Victorian Endowment for Science, Innovation and Technology. Apologies to authors whose work could not be cited due to space limitations. References [1] Lobo NA, Shimono Y, Qian D, Clarke MF. The biology of cancer stem cells. Annu Rev Cell Dev Biol 2007;23:675–99. [2] He S, Nakada D, Morrison SJ. Mechanisms of stem cell self-renewal. Annu Rev Cell Dev Biol 2009;25:377–406. [3] Dick JE. Stem cell concepts renew cancer research. Blood 2008;112:4793–807. [4] Shackleton M, Quintana E, Fearon ER, Morrison SJ. Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell 2009;138:822–9. [5] Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB. Tumourinitiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 2009;8:806–23. [6] Reya T, Morrison SJ, Clarke MF, Weissman IL. Stem cells, cancer, and cancer stem cells. Nature 2001;414:105–11. [7] Nowell PC. The clonal evolution of tumor cell populations. Science 1976;194:23–8. [8] Foulds L. The natural history of cancer. J Chronic Dis 1958;8:2–37. [9] Fearon ER, Vogelstein B. A genetic model for colorectal tumorigenesis. Cell 1990;61:759–67. [10] Lengauer C, Kinzler KW, Vogelstein B. Genetic instabilities in human cancers. Nature 1998;396:643–9.
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