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Cancer Stem Cells: The Other Face of Janus
FESTSCHRIFT ARTICLE
Cancer Stem Cells: The Other Face of Janus Sahil Mittal, MD, Randy Mifflin, PhD and Don W. Powell, MD
Abstract: Advances in the field of stem cell biology have provided renewed hopes that stem cells can be used to treat a wide range of genetic diseases and traumatic injuries. However, advances in the field of cancer cell biology have led to formulation of the cancer stem cell hypothesis, which posits that cancers arise from mutant stem cells. Further, this hypothesis proposes that these stem cells account for cancer recurrence, metastasis, and resistance to conventional treatments. Thus, although normal stem cells represent potential effective solutions to numerous clinical problems, when mutated, they may also represent the cause of many human malignancies. Key Indexing Terms: Gastrointestinal stem cells; Cancer stem cells; Colorectal cancer. [Am J Med Sci 2009;338(2):107–112.]
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ll cultures entertain the concept of duality of spirit. To the ancient Romans, this was manifested by the god Janus, a deity with 2 faces, capable of looking into the past as well as the future. Our first month of the year is named after Janus and the name embodies the idea of past problems as well as future hopes. The Comedy and Tragedy masks of the Greek theater, the Yin-Yang concepts of oriental culture, and the Physician Jekyll and Mr. Hyde of western society are other examples of such duality. One can compare the field of stem cells to this concept of duality because contained in the therapeutic promise of stem cells as potential treatments for diverse diseases is the idea that cancer stem cells (CSCs) may be the reason that we cannot readily cure cancer; ie, that CSCs may be the other face of Janus, the Tragedy mask, the Yang, or the Mr. Hyde. This article will briefly review the field of stem cells as potentially helpful therapeutic reagents and the current evidence that, contained in malignant tumors, are difficult to destroy tumor initiating cells, or CSCs.
THE PROMISE OF STEM CELLS Stem cells represent one of the most promising areas of science because of its potential for cell-based therapies to treat disease. This field is often referred to as regenerative or reparative medicine. Research on stem cells exploded in the early 1960s when Canadian scientists Ernest A. McCulloch and James E. Till showed that bone marrow cells injected into irradiated mice created visible nodules in the mice’s spleens, in proportion to the number of bone marrow cells injected. In later work, they demonstrated that each nodule had its origin from a single cell and these cells were capable of self-renewal.1,2 The ‘‘stemness’’ of these cells is regulated by joint efforts from intrinsic properties of these cells and extrinsic signals that emanate from a specialized microenvironment where these stem cells reside. This location is called the stem cell niche.3 To study the properties of stem cells, it is important to identify and From the Departments of Internal Medicine (SM, RM, DWP), and Neuroscience and Cell Biology (DWP), University of Texas Medical Branch, Galveston, Texas. This study was supported by the NIDDK (DK-55783). Correspondence: Don W. Powell, MD, Department of Internal Medicine, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0764 (E-mail: [email protected]).
isolate them. Stem cells are most commonly identified by staining for cell surface markers (eg, proteins such as CD antigens), exclusion of fluorescent dyes, such as Hoechst 3342 or rhodamine (which stains mitochondria). Another method is to label the DNA of cells with tritiated thymidine. During the subsequent cell division, somatic cells dilute the nonradioactive thymidine; however, stem cells tend to retain the label by means of asymmetric segregation of the old and new DNA strands.4 Although most of the currently used markers do not recognize functional stem cell activity, combinations (typically with 3 to 5 different markers) allow for the purification of near-homogenous populations of stem cells. A wide variety of factors critical for stem cell regulation have been identified, including cytokines, growth factors, transcription factors, and cell cycle regulators. The integration of these diverse components is accomplished by various stem cell signaling pathways. For example, activation of the Notch signaling pathway and its downstream target genes has been demonstrated to increase the self-renewal capacity of stem cells. Similarly, Wnt activation leads to the stabilization of cytoplasmic -catenin, which acts as a transcription coactivator for target genes that are responsible for proliferation of intestinal crypts in the gastrointestinal epithelium. Conversely, loss of Wnt signaling causes a dramatic reduction in the number of crypt cells and crypts.5 On the other hand, when transforming growth factor-beta family growth factors bind their receptors on stem cells, they activate the Smad pathway, leading to the transcription of genes, resulting in potent inhibition of stem cell growth. Positive and negative regulators ultimately balance the transition from quiescence to proliferation of these stem cells. Adult stem cells are found in various niches throughout the body, such as bone marrow, brain, liver, and skin. There they represent a mechanism for tissue maintenance, growth, and repair in later life. These adult stem cells were originally thought to be committed to regenerating only a very restricted set of cell lineages; however, it is becoming increasingly evident that such stem cells show considerably more plasticity. There are several types of adult stem cells (see below).
WHAT IS A STEM CELL? The definition of a stem cell requires that the cell possess 2 fundamental properties: Self-Renewal The process whereby at least 1 daughter cell of a dividing stem cell retains stem cell properties (Figure 1). Potency Under the right conditions, or given the right signals (growth factors, cytokines, and matrix), stem cells can differentiate into cells that are normally derived from any of the 3 germ layers (endoderm, mesoderm, and ectoderm). Such stem cells are deemed pluripotent stem cells. Cells that can differentiate into a limited number of mature cell types (eg, hematopoietic cells forming the different lineages of blood cells) are called multipotent stem cells.
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human tissues, including neural, cardiac, vascular, pancreatic, hepatic, and bone, and this technique and the generated cells have the potential to be used for replacement therapy. Human iPS cells might be an ideal cell source for cell therapy, given that the iPS cells can be derived from the patient to be treated, and thus would avoid immune rejection. The production of iPS cell lines from patients with various diseases also provides a new opportunity to understand disease pathogenesis at the molecular level and to develop novel and effective treatments.6 Adult Stem Cells FIGURE 1. Symmetric and asymmetric division of stem cells. A symmetric division refers to generation of 2 identical daughter cells needed for expanding the stem cell pool. In an asymmetric division, only 1 of the daughter cells retains stem cell properties, whereas the progenitor cell proceeds down the path of proliferation and differentiation.
TYPES OF STEM CELLS Embryonic Stem Cells Embryonic stem (ES) cells are derived from early-stage embryos by transferring the inner cell mass of the blastocyst into a plastic laboratory culture dish. Over the course of several days, these cells proliferate and begin to crowd the culture dish. Then they may be removed gently and plated into several fresh culture dishes. ES cells that have proliferated in cell culture for months without differentiating, and prove to be pluripotent and genetically normal, are referred to as ES cell lines.6 The process of somatic cell nuclear transfer can also generate ES cell lines. This entails the transfer of a nucleus obtained from a somatic cell into an enucleated egg. The resulting blastocyst will contain the genomic material of the donor. Another method is to reprogram somatic cells into an embryonic-like state, and these cells are known as induced pluripotent stem (iPS) cells. This process has gained popularity because it is totally independent of the availability of embryonic cells. iPS cells are created by the introduction of a defined and limited set of transcription factors into the somatic cells and by culturing these cells under specific ES cell conditions.6 The cell surface antigens most commonly used to identify human ES cells are the glycolipids stage specific embryonic antigen 3 and stage specific embryonic antigen 4 and the keratan sulfate antigens tumor rejection antigen-1-60 and tumor rejection antigen-1-81, germ cell tumor marker 2 and germ cell tumor marker 343, and protein antigens CD9 and CD90.7 When removed from the factors that maintain them as stem cells, ES cells will differentiate. Three general approaches have been used to initiate differentiation. With the first ES cells form 3-dimensional aggregates known as embryoid bodies. In the second method, ES cells are cultured directly on stromal cells (fibroblasts and myofibroblasts). The third protocol involves differentiating them on a monolayer of extracellular matrix proteins. For example, the potential of ES cells to develop into lymphocytic B cells was demonstrated by the coculture of ES cells with stromal cells in a medium containing lymphoid cytokines. The transfer of serum-derived embryoid bodies into a serum-free medium followed by treatment with fibroblast growth factors and certain other factors promotes maturation into endocrine cells. Many differentiation protocols have been optimized by changing the culture medium composition, altering the culture surface, or by genetic modification via specific gene insertion. Such techniques may produce many
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Hematopoietic Stem Cells The best sources of hematopoietic stem cells (HSCs) are bone marrow and mobilized peripheral blood, although HSCs can also be isolated from placental and umbilical cord tissue. In humans, the combination of positive selection markers for CD34, CD90 and negative selection for lineage marked cells (cell surface antigens used for immunophenotyping cells of a particular developmental lineage) can identify a near-homogeneous population of HSCs.8 Although the application of most classes of adult stem cells is in an early phase of clinical testing, HSCs have seen widespread clinical use. Currently, the main indications for HSC transplantation, more commonly known as bone marrow transplantation, are hematopoietic cancers (leukemia and lymphomas). Other indications include other causes of bone marrow failure, such as aplastic anemia, thalassemia, and sickle cell anemia. Traditionally, HSC transplantation is preceded by intensive chemotherapy that completely destroys the bone marrow, known as myeloablation. Recently developed nonmyeloablative preconditioning protocols have greatly reduced the risk of the procedure and have enhanced the practice of bone marrow transplantation. A phenomenon known as transplantinduced tolerance has been shown, wherein hosts whose immune and blood cell systems generated with HSCs transplanted from a given donor were permanently capable of accepting an organ transplant (such as heart) from the same donor. The use of nonmyeloablative regimens to prepare the host should enable physicians to withhold chronic immunosuppression usually used to prevent rejection and switch to protocols that combine HSC and organ transplant and use transplant-induced tolerance. This should eliminate both graft versus host disease and chronic immunosuppressionrelated complications.9,10 Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from several, perhaps most, tissues, although bone marrow is most often used. Human MSCs are characterized by the expression of variable levels of CD105, CD73, CD44, CD90, CD71 (transferrin receptor), the ganglioside GD2 and CD271, and absence of the hematopoietic markers.11 The use of stem cells for repair requires that the target organ be easily accessible to stem cell administration. Transplantation experiments have shown that bone marrow-derived MSCs can spread to many tissues after injection or following tissue injury. They seem to home preferentially to the site of injury, where they support functional recovery. MSCs have been used to treat children with severe osteogenesis imperfecta, resulting in fewer fractures and to help bone marrow recovery after high-dose chemotherapy. The immunosuppressive effects of MSCs support their use in acute graft versus host disease, where they inhibit donor T-cell reactivity to the histocompatiVolume 338, Number 2, August 2009
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FIGURE 2. The stem cell niche of human colon. Bone morphogenetic protein (BMP) signaling becomes more active as one moves up a colonic crypt. Wnt signaling is more prevalent at the base of each crypt where epithelial stem cells are located. Within the stem cell niche, intestinal subepithelial myofibroblasts (ISEMF) and smooth muscle cells (SMC) of the muscularis mucosae secrete BMP antagonists that further promote proliferation and inhibit differentiation (Reprinted with permission from Kosinski C, Li VS, Chan AS, et al. Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. Proc Natl Acad Sci USA 2007;104: 15418–15423).
bility antigens of the recipient. Their immunomodulatory role is also being explored in treatment for Crohn disease.12,13 MSCs transplanted into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice differentiate into the various types of cells which ordinarily form the HSC niche. In addition, they also preserve the HSC pool in the bone marrow by maintaining the HSCs in a quiescent state.14 MSCs are thought to modulate the immune response by inhibiting dendritic cell maturation and affecting their functional properties, resulting in an antiinflammatory phenotype. Alternatively, when MSCs are present in an inflammatory microenvironment, they seem to inhibit interferon-␣ secretion from TH1 (helper T cells type 1) and natural killer (NK) cells and increase interleukin-4 secretion from TH2 cells, thereby skewing the immune response from TH1 to TH2 phenotype.15 Neural Stem (NS) Cells Recent discoveries have violated the long-standing dogma that stem cells are not present in the adult mammalian brain. With the help of selective immunocytochemical markers for NS cells: eg, Nestin, an intermediate filament; Sox 1, a transcription factor; and CD133, it has been shown that NS cells are present in the olfactory bulb and the hippocampus. However, the poor regenerative capacity of the adult brain, despite the demonstrated presence of NS cells, may be attributed to the inhibitory factors present in the adult brain and the number of NS cells could be too small for effective repair. NS cells in culture may be induced to differentiate by growing them on laminin in the presence of serum. With this protocol, the predominant cell type developed is the astrocyte. In the presence of neurotrophic factors, such as nerve growth factors, the yield is predominantly neurons, and if platelet-derived growth factor is present in the medium, oligodendrocytes increase in number.16,17 © 2009 Lippincott Williams & Wilkins
The hope is that neural stem cells (NS cells) will replace the damaged neurons in human degenerative disorders, such as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, as well as after brain and spinal cord injuries from stroke and trauma. However, to date, it has not been demonstrated that the new axons of the neurons formed by stem cell transplantation can connect to their targets to reestablish circuitry. A more likely role for NS cell transplantation may be to provide support cells that supply neurotrophic factors that may reverse damage caused by injury and disease.18 –20 At present, 2 clinical trials involving NS cell transplantation are ongoing: 1 is aimed at providing the missing enzyme in Batten disease, a degenerative disorder of childhood. The other is the use of human NS cells in the treatment of stroke.21 Recently, several biotech firms have received regulatory authority to proceed with trials of fetal NS cells or ES cells to treat stroke, spinal injury, or Pelizaeus-Merzbacher disease, a recessive X-linked dysmyelinating disorder of the central nervous system.22 Epithelial Stem Cells Most epithelia must constantly replace damaged or dead cells throughout the life of the animal to sustain function. This renewal is driven by stem cells, which on activation generate proliferating progeny referred to as transit amplifying cells. In this review, we will focus on the intestinal epithelium to define the role of stem cells in maintaining epithelial tissue homeostasis. The small intestine is organized into crypt and villi and characterized by 4 differentiated cells—the absorptive enterocyte and the secretory cells: goblet, enteroendocrine, and Paneth cells. By labeling cells with tritiated thymidine and then following their fate, it can be shown that the stem cells and their progeny, transit amplifying cells, reside in a region near the base of the crypt (Figure 2). As they migrate out of their niche,
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they cease to proliferate and begin to differentiate into the different cell lineages, which make the mature villus.5 The modeling and remodeling of the intestinal epithelium depends on epithelial–mesenchymal crosstalk. Wnt, bone morphogenetic protein (BMP), and hedgehog (Hh) signaling mediate the information flow between the epithelium and the mesenchymal cells of the niche (Figure 2). The niche in the small intestine and colon is formed by the subepithelial sheath of activated fibroblasts, also called myofibroblasts.23,24 The BMP signaling pathway functions as a negative regulator of crypt proliferation. When the BMP receptor is knocked out, or when the BMP antagonists Gremlin or noggin is overexpressed, an expansion of the stem cell population occurs.25–27 A similar profile can be seen in humans in the juvenile polyposis syndrome, and almost half of these cases are accounted by mutations in the BMP signaling pathway.28,29 The expression of BMP in the subepithelium is positively regulated by hedgehog signaling from the epithelium to the mesenchyme.30 This leads to inhibition of ectopic crypts formation around an established crypt and ensures proper spacing between the crypts. Functional studies confirm that the Wnt/-catenin pathway constitutes the master switch between proliferation and differentiation of the epithelial cells. In the absence of Wnt signals, -catenin is degraded by a complex that contains adenomatous polyposis coli (APC). When the Wnt pathway is overactivated by APC mutation, or mutation of other Wnt components, crypts become enlarged and villi become smaller, as if the normal migration of the cells along the crypt-villus axis and the differentiation into the different cell lineages forming the villus has been blocked. Opposing gradients of Wnt and BMP signals are present along the crypt axis with Wnt signals being highest at the crypt base (Figure 2).31 The Notch pathway plays a central role in deciding the absorptive versus secretory fate of these dividing epithelial cells. Loss of function in notch signaling drives the cells to the secretory lineage. Taken together, these signals converge to generate the distinct features of stem cells, including self-renewal and proliferation, deregulation of which could result in a diseased state, such as malignancy. Cancer Stem Cells A tumor is the result of unregulated cellular proliferation. Cancer can be considered a disease of unregulated stem cell self-renewal. In normal tissue, stem cells are at the bottom of the differentiation tree and the mature cells at the other end of the spectrum. A similar functional hierarchy can be seen in tumors, where tumorigenic cells undergo self-renewal and aberrant differentiation, resulting in a heterogeneous population of cells. Turning on the switch for unlimited proliferation may be easier in a stem cell than in a differentiated cell. Moreover, stem cells have a long life and hence a greater opportunity to accumulate mutations. For these reasons, it is reasonable to assume that stem cells maybe the target of mutations and transformation in cancer genesis. The concept of the CSC was first proposed in liquid tumors (myeloma and leukemia) when experiments showed that only a small percentage (1%– 4%) of cancer cells were capable of extensive proliferation and could form colonies.32–34 Using a murine model of Helicobacter pylori infection, Houghton et al35 showed that during periods of chronic inflammation, gastric tumors can seemingly arise from circulating bone marrow-derived stem cells. Since then, this phenomenon suggesting the existence of CSCs has also been shown for other solid cancers. Experiments suggest that only 1 in 1000 to 5000 or 0.01% to 0.002% of solid tumor cells, including colorectal cancer, form colonies in soft agar or form ectopic cancers when
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FIGURE 3. The cancer stem cell hypothesis. Cancer stem cells are multipotent and can give rise to the diverse set of cells that make up a given tumor. Current treatments for cancer can considerably diminish tumor burden but may have a negligible effect on CSCs, which are later capable of driving tumor recurrence and regrowth. To truly cure cancer, it will be necessary to develop novel therapies that are cytotoxic to CSCs. Reprinted by permission from Macmillan Publishers Ltd: Nature Clinical Practice Neurology (Das S, Srikanth M, Kessler JA. Cancer stem cells and glioma. Nat Clin Pract Neuro 2008;4:427– 435).41 Copyright 2008.
injected into immunocompromised mice.36 – 40 Human colorectal cancer cells enriched for CD133 expression initiate tumors in immunocompromised NOD/SCID mice, whereas the CD133-negative fraction does not.39,40 These observations led to the hypothesis that only an exceedingly small percentage of cancer cells present in a neoplasm are tumorigenic and thus have the ability to metastasize or regenerate after surgical excision and either radiation or chemotherapy (Figure 3). Under this hypothesis, these cells could be considered as ‘‘cancer stem cells.’’ In the experimental setting, only these CSCs would be capable of growing in the situation where a cancer is excised from 1 host, the cells individually dispersed, and then a certain number of these cells injected into immunocompromised mice to see if new tumors form (self-renewal assay). A substantial body of evidence supports this hypothesis that malignant tumors are initiated and maintained by a small population of cells within a tumor, and these cells possess properties similar to normal adult stem cells. These properties include the ability to self-renew and generate differentiated progeny. When a normal stem cell divides, it can generate daughter cells with indefinite replication capacity (self-renewal) and progenitor cells with a finite capacity for replication. Thus, a stem cell population represents an inexhaustible pool of self-renewing cells capable of generating a continuous supply of differentiated progeny.42 However, unlike normal stem cells, CSCs lack homeostatic control, which makes cancer the intractable threat that it is.43 Although CSCs may share properties with their normal counterparts, they may not necessarily have their origin from mutant stem cells. Rather, it is possible that they may be derived from mutant cancer cells that have acquired certain characteristics of stem cells (Figure 4). Thus, CSCs may be more appropriately called ‘‘tumor-initiating cells.’’ However, recent studies using genetically engineered mice support the Volume 338, Number 2, August 2009
Cancer Stem Cells: The Other Face of Janus
the high incidence of mutations along these axes observed in human colorectal cancer.28,29,50 Patients with a single mutated copy of the APC gene contain an expansion of the stem cell compartment before any histologically visible neoplastic changes.51 Many of the same processes underlying maintenance of the normal epithelial stem cell niche are likely to also be important in maintaining the CSC niche. The CSC hypothesis suggests a rethinking of current clinical practices. If the CSCs are responsible for long-term recurrence, chemoresistance, and subsequent metastasis of tumors, then therapies must be developed to target the CSCs themselves to cure the disease.
CONCLUSIONS
FIGURE 4. Origin of cancer stem cells. Self-renewal and differentiation potentials are the features of stem cells. Progenitor cells, the product of stem cells that lose the activity of self-renewal, could differentiate into mature cells, which have the feature of differentiation. In theory, mutations in stem, progenitor, or terminally differentiated cells could create a tumor cell with stem-like properties. Recent data suggest that colorectal cancer is initiated by mutant stem cells (Reprinted with permission from BioMed Central Ltd. Ponnusamy MP, Batra SK. Ovarian cancer: emerging concept on cancer stem cells. J Ovarian Res 2008;1:4).44
notion that CSCs are indeed mutant intestinal epithelial stem cells.45,46 According to the CSC hypothesis, only a small subset of tumor cells should be able to initiate and sustain malignant tumor growth and give rise to the phenotypic heterogeneity observed in the original tumor (Figure 3). This has been found to be true for a wide variety of cancers, including colorectal cancer.39,40,43 Recently, Quintana et al,47 using a modified protocol, have demonstrated that in human melanomas, the frequency of tumor-initiating cells can be as high as 25% of tumor cells, a finding that challenges the CSC hypothesis. This relatively high number of tumor-initiating cells was discovered when melanoma cells were injected into mice (NOD/SCID mice that also have a molecular defect in the interleukin-2 receptor) that are even more profoundly immunocompromised than conventional NOD/SCID mice. This raises the question whether the ability to initiate tumors de novo also involves the ability of the injected cells to escape immune surveillance in addition to the tumorigenic potential of the injected cancer cells. Another possibility is that melanoma, one of the most aggressive and malignant of cancers, inherently processes more tumorigenic cells than do solid tumors. Similar to the regulation of normal intestinal epithelial stem cells, CSC proliferation is thought to be controlled by a yin-yang equilibrium in which Wnt signaling drives proliferation and BMP signaling counters proliferation.48,49 Wnt proteins may be secreted by both epithelial and mesenchymal cells, whereas BMPs come solely from the stromal compartment.23 Thus, alterations in myofibroblast Wnt or BMP signaling could lead to stem cell proliferation and hyperplastic or neoplastic progression. The importance of these pathways is evident from © 2009 Lippincott Williams & Wilkins
Before concluding that CSCs are the Mr. Hyde of the stem cell world, 2 caveats must be noted: first, perhaps, nonmalignant stem cell therapies will not be the panacea expected. Donor type leukemias are reported following HSC transplantation and donor-derived multifocal gliomas have recently been reported after unregulated neural stem cell transplantation.52 Perhaps, the other face of Janus can make its appearance during normal stem cells therapies. The other caveat is that the definition and understanding of CSCs, should the CSCs hypothesis prove true, would provide new targets for the successful treatment of cancer. Thus, the CSC hypothesis holds the promise of a cure for this dreaded disease, and the good face of Janus might arise from the CSC field. REFERENCES 1. Siminovitch L, McCulloch EA, Till JE. The distribution of colonyforming cells among spleen colonies. J Cell Physiol 1963;62:327–36. 2. Becker AJ, McCulloch EA, Till JE. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 1963;197:452– 4. 3. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7–25. 4. Potten CS, Owen G, Booth D. Intestinal stem cells protect their genome by selective segregation of template DNA strands. J Cell Sci 2002;115(pt 11):2381– 8. 5. van der Flier LG, Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol 2009;71:241– 60. 6. Yu J, Thomson JA. Pluripotent stem cell lines. Genes Dev 2008;22: 1987–97. 7. Adewumi O, Aflatoonian B, Ahrlund-Richter L, et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 2007;25:803–16. 8. Baum CM, Weissman IL, Tsukamoto AS, et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 1992;89:2804 – 8. 9. Sykes M. Hematopoietic cell transplantation for tolerance induction: animal models to clinical trials. Transplantation 2009;87:309 –16. 10. Buchmann I, Meyer RG, Mier W, et al. Myeloablative radioimmunotherapy in conditioning prior to haematological stem cell transplantation: closing the gap between benefit and toxicity? Eur J Nucl Med Mol Imaging 2009;36:484 –98. 11. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008;8:726 –36. 12. Mishra PJ, Glod JW, Banerjee D. Mesenchymal stem cells: flip side of the coin. Cancer Res 2009;69:1255– 8. 13. Karp JM, Leng Teo GS. Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell 2009;4:206 –16. 14. Muguruma Y, Yahata T, Miyatake H, et al. Reconstitution of the
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15. Aggarwal S, Pittenger MF. Human mesenchymal stem cells modulate allogeneic immune cell responses. Blood 2005;105:1815–22.
33. Bruce WR, Van Der Gaag H. A quantitative assay for the number of murine lymphoma cells capable of proliferation in vivo. Nature 1963; 199:79 – 80.
16. Micci MA, Pasricha PJ. Neural stem cells for the treatment of disorders of the enteric nervous system: strategies and challenges. Dev Dyn 2007;236:33– 43.
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17. Ahmed S. The culture of neural stem cells. J Cell Biochem 2009; 106:1– 6.
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19. Torrente Y, Polli E. Mesenchymal stem cell transplantation for neurodegenerative diseases. Cell Transplant 2008;17:1103–13. 20. Reimers D, Gonzalo-Gobernado R, Herranz AS, et al. Driving neural stem cells towards a desired phenotype. Curr Stem Cell Res Ther 2008;3:247–53. 21. Taupin P. HuCNS-SC (StemCells). Curr Opin Mol Ther 2006;8: 156 – 63. 22. Duncan ID. Oligodendrocytes and stem cell transplantation: their potential in the treatment of leukoencephalopathies. J Inherit Metab Dis 2005;28:357– 68. 23. Powell DW, Adegboyega PA, Di Mari JF, et al. Epithelial cells and their neighbors. I. Role of intestinal myofibroblasts in development, repair, and cancer. Am J Physiol Gastrointest Liver Physiol 2005;289: G2–7. 24. Powell DW, Mifflin RC, Valentich JD, et al. Myofibroblasts. II. Intestinal subepithelial myofibroblasts. Am J Physiol 1999;277(2 pt 1):C183–C201. 25. Sneddon JB, Zhen HH, Montgomery K, et al. Bone morphogenetic protein antagonist gremlin 1 is widely expressed by cancer-associated stromal cells and can promote tumor cell proliferation. PNAS 2006; 103:14842–7.
37. Singh SK, Hawkins C, Clarke ID, et al. Identification of human brain tumour initiating cells. Nature 2004;432:396 – 401. 38. Dalerba P, Dylla SJ, Park I-K, et al. Phenotypic characterization of human colorectal cancer stem cells. PNAS 2007;104:10158 – 63. 39. O’Brien CA, Pollett A, Gallinger S, et al. A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 2007;445:106 –10. 40. Ricci-Vitiani L, Lombardi DG, Pilozzi E, et al. Identification and expansion of human colon-cancer-initiating cells. Nature 2007;445: 111–5. 41. Das S, Srikanth M, Kessler JA. Cancer stem cells and glioma. Nat Clin Pract Neuro 2008;4:427–35. 42. Tan BT, Park CY, Ailles LE, et al. The cancer stem cell hypothesis: a work in progress. Lab Invest 2006;86:1203–7. 43. Dalerba P, Cho RW, Clarke MF. Cancer stem cells: models and concepts. Annu Rev Med 2007;58:267– 84. 44. Ponnusamy MP, Batra SK. Ovarian cancer: emerging concept on cancer stem cells. J Ovarian Res 2008;1:4. 45. Barker N, Ridgway RA, van Es JH, et al. Crypt stem cells as the cells-of-origin of intestinal cancer. Nature 2009;457:608 –11.
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Volume 338, Number 2, August 2009
Cancer Stem Cells: The Other Face of Janus Sahil Mittal, MD, Randy Mifflin, PhD and Don W. Powell, MD
Abstract: Advances in the field of stem cell biology have provided renewed hopes that stem cells can be used to treat a wide range of genetic diseases and traumatic injuries. However, advances in the field of cancer cell biology have led to formulation of the cancer stem cell hypothesis, which posits that cancers arise from mutant stem cells. Further, this hypothesis proposes that these stem cells account for cancer recurrence, metastasis, and resistance to conventional treatments. Thus, although normal stem cells represent potential effective solutions to numerous clinical problems, when mutated, they may also represent the cause of many human malignancies. Key Indexing Terms: Gastrointestinal stem cells; Cancer stem cells; Colorectal cancer. [Am J Med Sci 2009;338(2):107–112.]
A
ll cultures entertain the concept of duality of spirit. To the ancient Romans, this was manifested by the god Janus, a deity with 2 faces, capable of looking into the past as well as the future. Our first month of the year is named after Janus and the name embodies the idea of past problems as well as future hopes. The Comedy and Tragedy masks of the Greek theater, the Yin-Yang concepts of oriental culture, and the Physician Jekyll and Mr. Hyde of western society are other examples of such duality. One can compare the field of stem cells to this concept of duality because contained in the therapeutic promise of stem cells as potential treatments for diverse diseases is the idea that cancer stem cells (CSCs) may be the reason that we cannot readily cure cancer; ie, that CSCs may be the other face of Janus, the Tragedy mask, the Yang, or the Mr. Hyde. This article will briefly review the field of stem cells as potentially helpful therapeutic reagents and the current evidence that, contained in malignant tumors, are difficult to destroy tumor initiating cells, or CSCs.
THE PROMISE OF STEM CELLS Stem cells represent one of the most promising areas of science because of its potential for cell-based therapies to treat disease. This field is often referred to as regenerative or reparative medicine. Research on stem cells exploded in the early 1960s when Canadian scientists Ernest A. McCulloch and James E. Till showed that bone marrow cells injected into irradiated mice created visible nodules in the mice’s spleens, in proportion to the number of bone marrow cells injected. In later work, they demonstrated that each nodule had its origin from a single cell and these cells were capable of self-renewal.1,2 The ‘‘stemness’’ of these cells is regulated by joint efforts from intrinsic properties of these cells and extrinsic signals that emanate from a specialized microenvironment where these stem cells reside. This location is called the stem cell niche.3 To study the properties of stem cells, it is important to identify and From the Departments of Internal Medicine (SM, RM, DWP), and Neuroscience and Cell Biology (DWP), University of Texas Medical Branch, Galveston, Texas. This study was supported by the NIDDK (DK-55783). Correspondence: Don W. Powell, MD, Department of Internal Medicine, University of Texas Medical Branch, 301 University Blvd., Galveston, TX 77555-0764 (E-mail: [email protected]).
isolate them. Stem cells are most commonly identified by staining for cell surface markers (eg, proteins such as CD antigens), exclusion of fluorescent dyes, such as Hoechst 3342 or rhodamine (which stains mitochondria). Another method is to label the DNA of cells with tritiated thymidine. During the subsequent cell division, somatic cells dilute the nonradioactive thymidine; however, stem cells tend to retain the label by means of asymmetric segregation of the old and new DNA strands.4 Although most of the currently used markers do not recognize functional stem cell activity, combinations (typically with 3 to 5 different markers) allow for the purification of near-homogenous populations of stem cells. A wide variety of factors critical for stem cell regulation have been identified, including cytokines, growth factors, transcription factors, and cell cycle regulators. The integration of these diverse components is accomplished by various stem cell signaling pathways. For example, activation of the Notch signaling pathway and its downstream target genes has been demonstrated to increase the self-renewal capacity of stem cells. Similarly, Wnt activation leads to the stabilization of cytoplasmic -catenin, which acts as a transcription coactivator for target genes that are responsible for proliferation of intestinal crypts in the gastrointestinal epithelium. Conversely, loss of Wnt signaling causes a dramatic reduction in the number of crypt cells and crypts.5 On the other hand, when transforming growth factor-beta family growth factors bind their receptors on stem cells, they activate the Smad pathway, leading to the transcription of genes, resulting in potent inhibition of stem cell growth. Positive and negative regulators ultimately balance the transition from quiescence to proliferation of these stem cells. Adult stem cells are found in various niches throughout the body, such as bone marrow, brain, liver, and skin. There they represent a mechanism for tissue maintenance, growth, and repair in later life. These adult stem cells were originally thought to be committed to regenerating only a very restricted set of cell lineages; however, it is becoming increasingly evident that such stem cells show considerably more plasticity. There are several types of adult stem cells (see below).
WHAT IS A STEM CELL? The definition of a stem cell requires that the cell possess 2 fundamental properties: Self-Renewal The process whereby at least 1 daughter cell of a dividing stem cell retains stem cell properties (Figure 1). Potency Under the right conditions, or given the right signals (growth factors, cytokines, and matrix), stem cells can differentiate into cells that are normally derived from any of the 3 germ layers (endoderm, mesoderm, and ectoderm). Such stem cells are deemed pluripotent stem cells. Cells that can differentiate into a limited number of mature cell types (eg, hematopoietic cells forming the different lineages of blood cells) are called multipotent stem cells.
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human tissues, including neural, cardiac, vascular, pancreatic, hepatic, and bone, and this technique and the generated cells have the potential to be used for replacement therapy. Human iPS cells might be an ideal cell source for cell therapy, given that the iPS cells can be derived from the patient to be treated, and thus would avoid immune rejection. The production of iPS cell lines from patients with various diseases also provides a new opportunity to understand disease pathogenesis at the molecular level and to develop novel and effective treatments.6 Adult Stem Cells FIGURE 1. Symmetric and asymmetric division of stem cells. A symmetric division refers to generation of 2 identical daughter cells needed for expanding the stem cell pool. In an asymmetric division, only 1 of the daughter cells retains stem cell properties, whereas the progenitor cell proceeds down the path of proliferation and differentiation.
TYPES OF STEM CELLS Embryonic Stem Cells Embryonic stem (ES) cells are derived from early-stage embryos by transferring the inner cell mass of the blastocyst into a plastic laboratory culture dish. Over the course of several days, these cells proliferate and begin to crowd the culture dish. Then they may be removed gently and plated into several fresh culture dishes. ES cells that have proliferated in cell culture for months without differentiating, and prove to be pluripotent and genetically normal, are referred to as ES cell lines.6 The process of somatic cell nuclear transfer can also generate ES cell lines. This entails the transfer of a nucleus obtained from a somatic cell into an enucleated egg. The resulting blastocyst will contain the genomic material of the donor. Another method is to reprogram somatic cells into an embryonic-like state, and these cells are known as induced pluripotent stem (iPS) cells. This process has gained popularity because it is totally independent of the availability of embryonic cells. iPS cells are created by the introduction of a defined and limited set of transcription factors into the somatic cells and by culturing these cells under specific ES cell conditions.6 The cell surface antigens most commonly used to identify human ES cells are the glycolipids stage specific embryonic antigen 3 and stage specific embryonic antigen 4 and the keratan sulfate antigens tumor rejection antigen-1-60 and tumor rejection antigen-1-81, germ cell tumor marker 2 and germ cell tumor marker 343, and protein antigens CD9 and CD90.7 When removed from the factors that maintain them as stem cells, ES cells will differentiate. Three general approaches have been used to initiate differentiation. With the first ES cells form 3-dimensional aggregates known as embryoid bodies. In the second method, ES cells are cultured directly on stromal cells (fibroblasts and myofibroblasts). The third protocol involves differentiating them on a monolayer of extracellular matrix proteins. For example, the potential of ES cells to develop into lymphocytic B cells was demonstrated by the coculture of ES cells with stromal cells in a medium containing lymphoid cytokines. The transfer of serum-derived embryoid bodies into a serum-free medium followed by treatment with fibroblast growth factors and certain other factors promotes maturation into endocrine cells. Many differentiation protocols have been optimized by changing the culture medium composition, altering the culture surface, or by genetic modification via specific gene insertion. Such techniques may produce many
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Hematopoietic Stem Cells The best sources of hematopoietic stem cells (HSCs) are bone marrow and mobilized peripheral blood, although HSCs can also be isolated from placental and umbilical cord tissue. In humans, the combination of positive selection markers for CD34, CD90 and negative selection for lineage marked cells (cell surface antigens used for immunophenotyping cells of a particular developmental lineage) can identify a near-homogeneous population of HSCs.8 Although the application of most classes of adult stem cells is in an early phase of clinical testing, HSCs have seen widespread clinical use. Currently, the main indications for HSC transplantation, more commonly known as bone marrow transplantation, are hematopoietic cancers (leukemia and lymphomas). Other indications include other causes of bone marrow failure, such as aplastic anemia, thalassemia, and sickle cell anemia. Traditionally, HSC transplantation is preceded by intensive chemotherapy that completely destroys the bone marrow, known as myeloablation. Recently developed nonmyeloablative preconditioning protocols have greatly reduced the risk of the procedure and have enhanced the practice of bone marrow transplantation. A phenomenon known as transplantinduced tolerance has been shown, wherein hosts whose immune and blood cell systems generated with HSCs transplanted from a given donor were permanently capable of accepting an organ transplant (such as heart) from the same donor. The use of nonmyeloablative regimens to prepare the host should enable physicians to withhold chronic immunosuppression usually used to prevent rejection and switch to protocols that combine HSC and organ transplant and use transplant-induced tolerance. This should eliminate both graft versus host disease and chronic immunosuppressionrelated complications.9,10 Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) are a heterogeneous subset of stromal stem cells that can be isolated from several, perhaps most, tissues, although bone marrow is most often used. Human MSCs are characterized by the expression of variable levels of CD105, CD73, CD44, CD90, CD71 (transferrin receptor), the ganglioside GD2 and CD271, and absence of the hematopoietic markers.11 The use of stem cells for repair requires that the target organ be easily accessible to stem cell administration. Transplantation experiments have shown that bone marrow-derived MSCs can spread to many tissues after injection or following tissue injury. They seem to home preferentially to the site of injury, where they support functional recovery. MSCs have been used to treat children with severe osteogenesis imperfecta, resulting in fewer fractures and to help bone marrow recovery after high-dose chemotherapy. The immunosuppressive effects of MSCs support their use in acute graft versus host disease, where they inhibit donor T-cell reactivity to the histocompatiVolume 338, Number 2, August 2009
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FIGURE 2. The stem cell niche of human colon. Bone morphogenetic protein (BMP) signaling becomes more active as one moves up a colonic crypt. Wnt signaling is more prevalent at the base of each crypt where epithelial stem cells are located. Within the stem cell niche, intestinal subepithelial myofibroblasts (ISEMF) and smooth muscle cells (SMC) of the muscularis mucosae secrete BMP antagonists that further promote proliferation and inhibit differentiation (Reprinted with permission from Kosinski C, Li VS, Chan AS, et al. Gene expression patterns of human colon tops and basal crypts and BMP antagonists as intestinal stem cell niche factors. Proc Natl Acad Sci USA 2007;104: 15418–15423).
bility antigens of the recipient. Their immunomodulatory role is also being explored in treatment for Crohn disease.12,13 MSCs transplanted into nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice differentiate into the various types of cells which ordinarily form the HSC niche. In addition, they also preserve the HSC pool in the bone marrow by maintaining the HSCs in a quiescent state.14 MSCs are thought to modulate the immune response by inhibiting dendritic cell maturation and affecting their functional properties, resulting in an antiinflammatory phenotype. Alternatively, when MSCs are present in an inflammatory microenvironment, they seem to inhibit interferon-␣ secretion from TH1 (helper T cells type 1) and natural killer (NK) cells and increase interleukin-4 secretion from TH2 cells, thereby skewing the immune response from TH1 to TH2 phenotype.15 Neural Stem (NS) Cells Recent discoveries have violated the long-standing dogma that stem cells are not present in the adult mammalian brain. With the help of selective immunocytochemical markers for NS cells: eg, Nestin, an intermediate filament; Sox 1, a transcription factor; and CD133, it has been shown that NS cells are present in the olfactory bulb and the hippocampus. However, the poor regenerative capacity of the adult brain, despite the demonstrated presence of NS cells, may be attributed to the inhibitory factors present in the adult brain and the number of NS cells could be too small for effective repair. NS cells in culture may be induced to differentiate by growing them on laminin in the presence of serum. With this protocol, the predominant cell type developed is the astrocyte. In the presence of neurotrophic factors, such as nerve growth factors, the yield is predominantly neurons, and if platelet-derived growth factor is present in the medium, oligodendrocytes increase in number.16,17 © 2009 Lippincott Williams & Wilkins
The hope is that neural stem cells (NS cells) will replace the damaged neurons in human degenerative disorders, such as Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, as well as after brain and spinal cord injuries from stroke and trauma. However, to date, it has not been demonstrated that the new axons of the neurons formed by stem cell transplantation can connect to their targets to reestablish circuitry. A more likely role for NS cell transplantation may be to provide support cells that supply neurotrophic factors that may reverse damage caused by injury and disease.18 –20 At present, 2 clinical trials involving NS cell transplantation are ongoing: 1 is aimed at providing the missing enzyme in Batten disease, a degenerative disorder of childhood. The other is the use of human NS cells in the treatment of stroke.21 Recently, several biotech firms have received regulatory authority to proceed with trials of fetal NS cells or ES cells to treat stroke, spinal injury, or Pelizaeus-Merzbacher disease, a recessive X-linked dysmyelinating disorder of the central nervous system.22 Epithelial Stem Cells Most epithelia must constantly replace damaged or dead cells throughout the life of the animal to sustain function. This renewal is driven by stem cells, which on activation generate proliferating progeny referred to as transit amplifying cells. In this review, we will focus on the intestinal epithelium to define the role of stem cells in maintaining epithelial tissue homeostasis. The small intestine is organized into crypt and villi and characterized by 4 differentiated cells—the absorptive enterocyte and the secretory cells: goblet, enteroendocrine, and Paneth cells. By labeling cells with tritiated thymidine and then following their fate, it can be shown that the stem cells and their progeny, transit amplifying cells, reside in a region near the base of the crypt (Figure 2). As they migrate out of their niche,
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they cease to proliferate and begin to differentiate into the different cell lineages, which make the mature villus.5 The modeling and remodeling of the intestinal epithelium depends on epithelial–mesenchymal crosstalk. Wnt, bone morphogenetic protein (BMP), and hedgehog (Hh) signaling mediate the information flow between the epithelium and the mesenchymal cells of the niche (Figure 2). The niche in the small intestine and colon is formed by the subepithelial sheath of activated fibroblasts, also called myofibroblasts.23,24 The BMP signaling pathway functions as a negative regulator of crypt proliferation. When the BMP receptor is knocked out, or when the BMP antagonists Gremlin or noggin is overexpressed, an expansion of the stem cell population occurs.25–27 A similar profile can be seen in humans in the juvenile polyposis syndrome, and almost half of these cases are accounted by mutations in the BMP signaling pathway.28,29 The expression of BMP in the subepithelium is positively regulated by hedgehog signaling from the epithelium to the mesenchyme.30 This leads to inhibition of ectopic crypts formation around an established crypt and ensures proper spacing between the crypts. Functional studies confirm that the Wnt/-catenin pathway constitutes the master switch between proliferation and differentiation of the epithelial cells. In the absence of Wnt signals, -catenin is degraded by a complex that contains adenomatous polyposis coli (APC). When the Wnt pathway is overactivated by APC mutation, or mutation of other Wnt components, crypts become enlarged and villi become smaller, as if the normal migration of the cells along the crypt-villus axis and the differentiation into the different cell lineages forming the villus has been blocked. Opposing gradients of Wnt and BMP signals are present along the crypt axis with Wnt signals being highest at the crypt base (Figure 2).31 The Notch pathway plays a central role in deciding the absorptive versus secretory fate of these dividing epithelial cells. Loss of function in notch signaling drives the cells to the secretory lineage. Taken together, these signals converge to generate the distinct features of stem cells, including self-renewal and proliferation, deregulation of which could result in a diseased state, such as malignancy. Cancer Stem Cells A tumor is the result of unregulated cellular proliferation. Cancer can be considered a disease of unregulated stem cell self-renewal. In normal tissue, stem cells are at the bottom of the differentiation tree and the mature cells at the other end of the spectrum. A similar functional hierarchy can be seen in tumors, where tumorigenic cells undergo self-renewal and aberrant differentiation, resulting in a heterogeneous population of cells. Turning on the switch for unlimited proliferation may be easier in a stem cell than in a differentiated cell. Moreover, stem cells have a long life and hence a greater opportunity to accumulate mutations. For these reasons, it is reasonable to assume that stem cells maybe the target of mutations and transformation in cancer genesis. The concept of the CSC was first proposed in liquid tumors (myeloma and leukemia) when experiments showed that only a small percentage (1%– 4%) of cancer cells were capable of extensive proliferation and could form colonies.32–34 Using a murine model of Helicobacter pylori infection, Houghton et al35 showed that during periods of chronic inflammation, gastric tumors can seemingly arise from circulating bone marrow-derived stem cells. Since then, this phenomenon suggesting the existence of CSCs has also been shown for other solid cancers. Experiments suggest that only 1 in 1000 to 5000 or 0.01% to 0.002% of solid tumor cells, including colorectal cancer, form colonies in soft agar or form ectopic cancers when
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FIGURE 3. The cancer stem cell hypothesis. Cancer stem cells are multipotent and can give rise to the diverse set of cells that make up a given tumor. Current treatments for cancer can considerably diminish tumor burden but may have a negligible effect on CSCs, which are later capable of driving tumor recurrence and regrowth. To truly cure cancer, it will be necessary to develop novel therapies that are cytotoxic to CSCs. Reprinted by permission from Macmillan Publishers Ltd: Nature Clinical Practice Neurology (Das S, Srikanth M, Kessler JA. Cancer stem cells and glioma. Nat Clin Pract Neuro 2008;4:427– 435).41 Copyright 2008.
injected into immunocompromised mice.36 – 40 Human colorectal cancer cells enriched for CD133 expression initiate tumors in immunocompromised NOD/SCID mice, whereas the CD133-negative fraction does not.39,40 These observations led to the hypothesis that only an exceedingly small percentage of cancer cells present in a neoplasm are tumorigenic and thus have the ability to metastasize or regenerate after surgical excision and either radiation or chemotherapy (Figure 3). Under this hypothesis, these cells could be considered as ‘‘cancer stem cells.’’ In the experimental setting, only these CSCs would be capable of growing in the situation where a cancer is excised from 1 host, the cells individually dispersed, and then a certain number of these cells injected into immunocompromised mice to see if new tumors form (self-renewal assay). A substantial body of evidence supports this hypothesis that malignant tumors are initiated and maintained by a small population of cells within a tumor, and these cells possess properties similar to normal adult stem cells. These properties include the ability to self-renew and generate differentiated progeny. When a normal stem cell divides, it can generate daughter cells with indefinite replication capacity (self-renewal) and progenitor cells with a finite capacity for replication. Thus, a stem cell population represents an inexhaustible pool of self-renewing cells capable of generating a continuous supply of differentiated progeny.42 However, unlike normal stem cells, CSCs lack homeostatic control, which makes cancer the intractable threat that it is.43 Although CSCs may share properties with their normal counterparts, they may not necessarily have their origin from mutant stem cells. Rather, it is possible that they may be derived from mutant cancer cells that have acquired certain characteristics of stem cells (Figure 4). Thus, CSCs may be more appropriately called ‘‘tumor-initiating cells.’’ However, recent studies using genetically engineered mice support the Volume 338, Number 2, August 2009
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the high incidence of mutations along these axes observed in human colorectal cancer.28,29,50 Patients with a single mutated copy of the APC gene contain an expansion of the stem cell compartment before any histologically visible neoplastic changes.51 Many of the same processes underlying maintenance of the normal epithelial stem cell niche are likely to also be important in maintaining the CSC niche. The CSC hypothesis suggests a rethinking of current clinical practices. If the CSCs are responsible for long-term recurrence, chemoresistance, and subsequent metastasis of tumors, then therapies must be developed to target the CSCs themselves to cure the disease.
CONCLUSIONS
FIGURE 4. Origin of cancer stem cells. Self-renewal and differentiation potentials are the features of stem cells. Progenitor cells, the product of stem cells that lose the activity of self-renewal, could differentiate into mature cells, which have the feature of differentiation. In theory, mutations in stem, progenitor, or terminally differentiated cells could create a tumor cell with stem-like properties. Recent data suggest that colorectal cancer is initiated by mutant stem cells (Reprinted with permission from BioMed Central Ltd. Ponnusamy MP, Batra SK. Ovarian cancer: emerging concept on cancer stem cells. J Ovarian Res 2008;1:4).44
notion that CSCs are indeed mutant intestinal epithelial stem cells.45,46 According to the CSC hypothesis, only a small subset of tumor cells should be able to initiate and sustain malignant tumor growth and give rise to the phenotypic heterogeneity observed in the original tumor (Figure 3). This has been found to be true for a wide variety of cancers, including colorectal cancer.39,40,43 Recently, Quintana et al,47 using a modified protocol, have demonstrated that in human melanomas, the frequency of tumor-initiating cells can be as high as 25% of tumor cells, a finding that challenges the CSC hypothesis. This relatively high number of tumor-initiating cells was discovered when melanoma cells were injected into mice (NOD/SCID mice that also have a molecular defect in the interleukin-2 receptor) that are even more profoundly immunocompromised than conventional NOD/SCID mice. This raises the question whether the ability to initiate tumors de novo also involves the ability of the injected cells to escape immune surveillance in addition to the tumorigenic potential of the injected cancer cells. Another possibility is that melanoma, one of the most aggressive and malignant of cancers, inherently processes more tumorigenic cells than do solid tumors. Similar to the regulation of normal intestinal epithelial stem cells, CSC proliferation is thought to be controlled by a yin-yang equilibrium in which Wnt signaling drives proliferation and BMP signaling counters proliferation.48,49 Wnt proteins may be secreted by both epithelial and mesenchymal cells, whereas BMPs come solely from the stromal compartment.23 Thus, alterations in myofibroblast Wnt or BMP signaling could lead to stem cell proliferation and hyperplastic or neoplastic progression. The importance of these pathways is evident from © 2009 Lippincott Williams & Wilkins
Before concluding that CSCs are the Mr. Hyde of the stem cell world, 2 caveats must be noted: first, perhaps, nonmalignant stem cell therapies will not be the panacea expected. Donor type leukemias are reported following HSC transplantation and donor-derived multifocal gliomas have recently been reported after unregulated neural stem cell transplantation.52 Perhaps, the other face of Janus can make its appearance during normal stem cells therapies. The other caveat is that the definition and understanding of CSCs, should the CSCs hypothesis prove true, would provide new targets for the successful treatment of cancer. Thus, the CSC hypothesis holds the promise of a cure for this dreaded disease, and the good face of Janus might arise from the CSC field. REFERENCES 1. Siminovitch L, McCulloch EA, Till JE. The distribution of colonyforming cells among spleen colonies. J Cell Physiol 1963;62:327–36. 2. Becker AJ, McCulloch EA, Till JE. Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 1963;197:452– 4. 3. Schofield R. The relationship between the spleen colony-forming cell and the haemopoietic stem cell. Blood Cells 1978;4:7–25. 4. Potten CS, Owen G, Booth D. Intestinal stem cells protect their genome by selective segregation of template DNA strands. J Cell Sci 2002;115(pt 11):2381– 8. 5. van der Flier LG, Clevers H. Stem cells, self-renewal, and differentiation in the intestinal epithelium. Annu Rev Physiol 2009;71:241– 60. 6. Yu J, Thomson JA. Pluripotent stem cell lines. Genes Dev 2008;22: 1987–97. 7. Adewumi O, Aflatoonian B, Ahrlund-Richter L, et al. Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nat Biotechnol 2007;25:803–16. 8. Baum CM, Weissman IL, Tsukamoto AS, et al. Isolation of a candidate human hematopoietic stem-cell population. Proc Natl Acad Sci USA 1992;89:2804 – 8. 9. Sykes M. Hematopoietic cell transplantation for tolerance induction: animal models to clinical trials. Transplantation 2009;87:309 –16. 10. Buchmann I, Meyer RG, Mier W, et al. Myeloablative radioimmunotherapy in conditioning prior to haematological stem cell transplantation: closing the gap between benefit and toxicity? Eur J Nucl Med Mol Imaging 2009;36:484 –98. 11. Uccelli A, Moretta L, Pistoia V. Mesenchymal stem cells in health and disease. Nat Rev Immunol 2008;8:726 –36. 12. Mishra PJ, Glod JW, Banerjee D. Mesenchymal stem cells: flip side of the coin. Cancer Res 2009;69:1255– 8. 13. Karp JM, Leng Teo GS. Mesenchymal stem cell homing: the devil is in the details. Cell Stem Cell 2009;4:206 –16. 14. Muguruma Y, Yahata T, Miyatake H, et al. Reconstitution of the
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