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Published as doi: 10.1096/fj.07-8560rev.
 (The FASEB Journal. 2007;21:3777-3785.)
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Cancer stem cell: target for anti-cancer therapy

Carol Tang*,{dagger},1, Beng T. Ang{ddagger},§ and Shazib Pervaiz{dagger},||,¶,#,1

* Department of Research,

{ddagger} Department of Neurosurgery, National Neuroscience Institute, Singapore;

{dagger} Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore;

§ Singapore Institute for Clinical Sciences, Agency for Science, Technology and Research, Singapore;

|| NUS Graduate School for Integrative Sciences and Engineering, National University of Singapore, Singapore;

Singapore-MIT Alliance, Singapore; and

# Duke-NUS Graduate Medical School, Singapore


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 IDENTIFICATION OF CANCER STEM...
 CHEMORESISTANCE
 RADIORESISTANCE
 INVOLVEMENT OF REACTIVE OXYGEN...
 CLINICAL IMPLICATIONS OF CANCER...
 REFERENCES
 
Cancer has long been viewed as a heterogeneous population of cells. While the great majority of cells that make up tumors are destined to differentiate, albeit aberrantly, and eventually stop dividing, only a minority population of cells, termed cancer stem cells, possess extensive self-renewal capability and can recapitulate tumor pathophysiology in an immune-compromised animal model. Tumor-initiating cells have been identified and isolated in a variety of cancers of the blood, breast, central nervous system, pancreas, skin, head and neck, colon, and prostate. In this review we present scientific evidence supporting the cancer stem cell model of tumor progression, and discuss the experimental and therapeutic implications. The concept of cancer stem cells may have profound implications for our understanding of tumor biology and for the design of novel treatments targeted toward these cells. Current therapeutic strategies include targeting the cancer stem cell as well as its microenvironmental niche. We present an interesting, novel strategy that takes into account the reactive oxygen species status in cancer stem cells and how it might serve as a method for eradicating these cells in tumor growth.—Tang, C., Ang, B. T., Pervaiz, S. Cancer stem cell: target for anti-cancer therapy.


Key Words: tumor stem cell • side population • CD133 • ROS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
IDENTIFICATION OF CANCER STEM...
 CHEMORESISTANCE
 RADIORESISTANCE
 INVOLVEMENT OF REACTIVE OXYGEN...
 CLINICAL IMPLICATIONS OF CANCER...
 REFERENCES
 
OVER THE LAST DECADE, IMPROVEMENTS in cancer therapies have prolonged the lives of cancer patients. However, after apparent successful initial therapy and recovery, development of secondary tumors often leads to a relapse of the disease. Emerging evidence has suggested that the capability of a tumor to grow and propagate is dependent on a small subset of cells within the tumor, termed cancer stem cells (CSCs). CSCs have now been identified and isolated in tumors of the hematopoietic system, breast, brain, prostate, colon, head and neck, and pancreas (Table 1 ) (1 2 3 4 5 6 7 8 9) . This subpopulation of CSCs is able to self-renew, differentiate, and regenerate a phenocopy of the original tumor when implanted into the nonobese diabetic, severe-combined immunodeficient (NOD-SCID) mouse. We present the current scientific evidence supporting the CSC hypothesis and discuss the experimental and therapeutic implications of the discovery of human CSCs. The concept of CSCs may have profound implications for our understanding of tumor biology and for the design of novel treatments targeted toward these cells for the complete eradication of tumor growth.


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  		Table 1. Cell surface markers associated with cancer stem cells


   IDENTIFICATION OF CANCER STEM CELLS
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF CANCER STEM...
CHEMORESISTANCE
 RADIORESISTANCE
 INVOLVEMENT OF REACTIVE OXYGEN...
 CLINICAL IMPLICATIONS OF CANCER...
 REFERENCES
 
For more than a century, human cancers have been recognized as a morphologically heterogeneous population of cells (10) . What has become clear in the past 10 years is that these cells are also functionally heterogeneous. Specifically, only a minority of tumor cells have the capacity to regenerate the tumor and sustain its growth when injected into an immune-compromised mouse model. At least two models of cancer growth can explain tumor development. The first, termed the stochastic model, assumes that every cancerous cell has the capacity to extensively proliferate and regenerate a tumor. This model predicts that isolating discrete populations of cells vs. the bulk population of cells on the basis of functional or phenotypic characteristics cannot enrich the tumor-initiating capacity. This is due to the assumption that all cancer cells have an equal, albeit low, probability of regenerating a tumor. In contrast, the cancer stem cell model assumes that only a very small subset of cells within the tumor population actually has the capacity to initiate and sustain tumor growth. By inference, a functional heterogeneity exists within the pool of cells that make up a tumor, and suggests that isolating and purifying this small population can generate a highly potent population of tumor-initiating cells.

The establishment of an in vivo model to test the tumor-initiating ability of discrete cellular populations, coupled with experimental methods of prospective purification of tumor cells, has allowed the identification of cancer stem cells from human leukemia (2) . Since the definition of the hematopoietic stem cell (HSC) by Till and McCulloch, landmark changes occurred in the late 1980s and early 1990s, when the distinct cell surface marker profile that would allow for the prospective isolation of normal HSCs by fluorescence-activated cell sorting (FACS) became known (11) . These advances led to the isolation of HSCs as well as multipotent and oligopotent progenitors that generate all mature blood cells. With the knowledge of the cell surface phenotypes for HSC and progenitor cells, investigators now had the ability to isolate similar subpopulations from acute myeloid leukemia (AML). John Dick and colleagues isolated and identified CD34+CD38– leukemic stem cells (LSCs) from human AML by FACS and demonstrated that these cells initiated leukemia in NOD-SCID mice compared with the CD34+CD38+ and CD34– fractions (2) . An engrafted leukemia could be serially transplanted into secondary recipients, providing functional evidence for self-renewal. Xenotransplantation, followed by serial transplantation, is now regarded as an essential criterion in defining cancer stem cells. The ability to recapture tumor pathophysiology is an important defining functional criterion of cancer stem cells prospectively isolated (Fig. 1 ).


Figure 1
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  		Figure 1. The cancer stem cell. Cell populations from leukemia are sorted by FACS and implanted at defined numbers in NOD-SCID mice. Only the fraction enriched in tumor-initiating cells would form serially transplantable tumors (continuation of red patch on flank of secondary mouse). The tumor xenograft established would be similar in morphology to the original patient’s tumor from which the cells were isolated.

In the last 5 years, investigation of solid tumor stem cells has gained momentum. Using similar approaches and principles of serial dilution and serial transplantation, solid tumor stem cells have been prospectively identified from a variety of cancers. In breast cancer, a minor, phenotypically distinct tumor cell population has been isolated that is able to form mammary tumors in NOD-SCID mice, whereas cells with alternative phenotypes are nontumorigenic even when implanted at significantly higher cell numbers, thereby demonstrating enrichment of tumor-initiating cells in selected fractions. The tumorigenic cells can be serially passaged, demonstrating self-renewal capacity, and are able to generate tumor heterogeneity, producing differentiated, nontumorigenic progeny. Thus, like AML, breast cancer growth appears to be driven by a rare population of tumor-initiating cells.

Investigators recently demonstrated the existence of CD133+ cells in human brain cancers that possess differentiative and self-renewal capacities and can initiate tumor growth in vivo, whereas CD133– cells cannot (9) . CD133+ cells made up the minority of cultured tumor cells, ranging from 3.5 to 46% of the total population; in general, increased CD133+ percentage was correlated with higher tumor grade. In vivo, injection of as few as 100 CD133+ cells could regenerate a serially transplantable phenocopy of the original tumor in the brains of NOD-SCID mice. This result was also obtained with cells isolated from pediatric medulloblastomas and from both pediatric and adult glioblastomas, raising the possibility that a common brain tumor stem cell exists in both adult and pediatric brain tumors. In contrast, injection of a 1000-fold higher CD133– cell number failed to elicit formation of tumor in all mice examined. Taylor et al. analyzed human ependymomas and subsequently demonstrated that purified CD133+ ependymoma cells showed tumorigenic ability in vivo, which was not observed in the more numerous CD133– population (12) . Taken together, these data indicate that the self-renewing and highly tumorigenic human brain tumor stem cell resides within the CD133+ population.


   CHEMORESISTANCE
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF CANCER STEM...
 CHEMORESISTANCE
RADIORESISTANCE
 INVOLVEMENT OF REACTIVE OXYGEN...
 CLINICAL IMPLICATIONS OF CANCER...
 REFERENCES
 
Cancer treatment has traditionally been based on the implicit assumption that human cancer populations are homogeneous. Cancer is resilient to treatment because malignant cells survive chemotherapy and radiation or avoid immune surveillance of endogenous cytotoxic T cells and natural killer cells. Cancer stem cells have a capacity for unlimited self-renewal, as well as the ability to initiate and drive tumor progression in an animal model (1 2 3 4 5 6 7 8 9) . Thus, they would seem the most probable candidates responsible for tumor chemoresistance and recurrence.

The "side population" (SP) is defined by Hoechst dye exclusion in flow cytometry and has been commonly used as one of the methods of enriching for cancer stem cells in a selected number of cancer cell lines and primary tumor cultures (13 14 15) . Goodell et al. have demonstrated that the exclusion of Hoechst 33342 dye by SP cells is a dynamic process involving the multidrug resistance transporter 1 (MDR1), a member of the ABC transporter transmembrane proteins (16) . However, MDR1 cannot be taken as a single marker to identify and isolate SP cells, and additional transporters should be analyzed. For instance, Zhou et al. have identified the BCRP1 multidrug resistance transporter to be the molecular determinant of the SP in mouse hematopoietic stem cells (17) .

Hirschmann-Jax and colleagues demonstrated that the SP of neuroblastoma cells not only had the characteristics of tumor stem cells (multipotentiality and self-renewal), but was also more resistant to the effects of drugs such as mitoxantrone and may contribute to the overall drug resistance phenotype of relapsed or resistant cancers (14) . Neuroblastoma cells cultured in the presence of mitoxantrone showed a progressive increase in SP cell frequency, indicating that their ability to expel mitoxantrone offered a survival advantage to these putative stem cells. Sorted SP cells, unlike non-SP cells, were also able to proliferate and establish new colonies in the presence of mitoxantrone, whereas non-SP cells could not, indicative of stem cell-like properties. Indeed, work by Szotek and colleagues has indicated that a similar SP stem cell-like population exists in genetically engineered mouse ovarian cancer cells as well as in human ovarian cancer cell lines (18) . This SP fraction could not be inhibited by the chemotherapeutic agent doxorubicin, but was inhibited, like non-SP cells, in the presence of Mullerian-inhibiting substance (MIS), suggesting that MIS signaling pathway transduction molecules exist in both SP and non-SP cells. This SP fraction also correlated with a consistent BCRP1 immunostaining pattern compared with non-SP cells. Taken together, the data demonstrate the link between SP and drug resistance, disease persistence, and relapse. Relapse following treatment with anticancer drugs is known to be a multifactorial problem. These studies highlight the significance of drug-resistant tumor stem cells. Work by our group has also shown that SP cells in human malignant glioma cell lines and primary glioblastoma multiforme (GBM) neurosphere cultures increased in the presence of temozolomide treatment, and that this SP fraction correlated with stem cell-like activity compared with the non-SP fraction (Fig. 2 , C. Chua et al., unpublished observations).


Figure 2
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  		Figure 2. Chemoresistance of SP stem-like cells. A) Three different glioma cell lines (U87MG, T98G, and U251) were treated with 100 µM temozolomide for 3 days. SP fold change is represented in the graph. P < 0.05 were taken to be statistically significant. B) After 3 days of temozolomide treatment, U87MG cells were sorted into SP and non-SP fractions, then subjected to immunostaining with ABCG2 and musashi-1, markers typically highly expressed in stem-like cells. The SP fraction demonstrated increased stem cell-like activity (P<0.05).

Much work has been carried out on brain tumor stem cells enriched by another marker, CD133. It is unclear at this time whether the SP overlaps with the CD133 population, but both markers have been shown to be highly enriched in neurosphere-forming capacity (19 , 20) , one of the defining characteristics of neural stem cells and progenitors (Fig. 3 ). However, because the neurosphere assay allows for expansion of both stem cells and progenitors, one must be diligent in assuring that a bona fide stem cell has been isolated by minimally demonstrating self-renewal over an extended period (>5 passages) coincident with the generation of a large number of progeny (21 , 22) . Owing to the erroneous belief that every sphere arises from a single neural stem cell, one must be careful when interpreting a neurosphere assay because it does not provide an accurate indication of stem cell frequency within.


Figure 3
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  		Figure 3. The neurosphere assay. The tumor specimen was disaggregated in trypsin dissociation medium, cleared of blood cells, and plated at clonal densities. The cells were cultured in serum-free growth medium in the presence of EGF and bFGF. Neurospheres were routinely passaged by mechanical trituration through a flame-drawn glass Pasteur pipette.

Liu and colleagues demonstrated for the first time an increased resistance of CD133+ brain tumor stem cells in response to treatment with chemotherapeutic agents such as temozolomide, carboplatin, paclitaxel (Taxol), and etoposide (VP16) compared with autologous CD133– cells (23) . Gene expression studies revealed a higher expression of multidrug resistance gene BCRP1 and DNA mismatch repair genes such as MGMT, as well as genes that inhibited apoptosis in the CD133-expressing cancer stem cells. These included antiapoptotic genes such as FLIP, BCL-2, and BCL-XL. The inhibitor of apoptosis protein family (IAP) genes such as XIAP, cIAP1, cIAP2, NAIP, and survivin were also found at higher expression levels in CD133+ cells. These results were consistent with and support the results of chemoresistance, in which CD133+ cells showed significant resistance to four common chemotherapeutic drugs compared with autologous CD133– cells. The work also showed that CD133 gene expression was significantly higher in recurrent GBM tissue specimens compared with their respective newly diagnosed tumors. It is observed clinically that tumors respond to chemotherapies only to recur with renewed resilience and aggression. Although chemotherapy kills most of the cells in a tumor, these results suggest that cancer stem cells may be left behind, which then recur due to their enhanced chemoresistance.

A separate study by Fan et al. implicated the Notch signaling pathway in embryonal brain tumors such as medulloblastoma (24) . The Notch signaling pathway is required in both non-neoplastic neural stem cells and embryonal brain tumors. Using pharmacologic inhibitors of Notch, the authors were able to show depletion of the specific brain tumor stem cell population defined by the CD133 marker or an ability to efflux the Hoechst 33342 dye, the SP. Notch was also expressed more highly in the stem cell-like fraction, providing a potential mechanism for their increased sensitivity to inhibition of this pathway. This depletion of stem cell fraction resulted in a loss of tumor-forming capacity. Apoptotic rates following Notch blockade were increased almost 10-fold in primitive nestin-positive, stem-like cells compared with nestin-negative cells. Taken together, the data imply that stem-like cells in medulloblastomas seemed to be selectively vulnerable to agents inhibiting the Notch pathway, thereby identifying a novel therapeutic target.


   RADIORESISTANCE
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF CANCER STEM...
 CHEMORESISTANCE
 RADIORESISTANCE
INVOLVEMENT OF REACTIVE OXYGEN...
 CLINICAL IMPLICATIONS OF CANCER...
 REFERENCES
 
Recent investigations in the field of brain and breast cancers implicate cancer stem cells in radiation resistance (25 26 27) . Bao et al. demonstrated that radiation resistance in highly malignant gliomas (GBM) is most likely mediated by tumor stem cells within (25) . They showed that CD133+ cancer stem cells contributed to glioma resistance through preferential activation of DNA damage checkpoint response and an increase in DNA repair capacity compared with CD133– tumor cells. The radioresistance of CD133+ glioma stem cells could be reversed with a specific inhibitor of Chk1 and Chk2 checkpoint kinases, which are closely associated with cellular resistance to radiation, thereby providing a therapeutic advantage to reducing brain tumor occurrence. As the cell cycle of a normal stem cell is tightly controlled by the checkpoint to maintain genomic stability and integrity, the defective checkpoint responses associated with early cancer development (28 , 29) implicate abnormal checkpoint control as a potential contributor to the transformation of normal cells into cancer stem cells.

Using conditions previously applied for culturing mammospheres from primary breast cancer specimens (30) to culture nonadherent cells that were isolated from two adherent breast cancer cell lines, Phillips et al. showed that cells arising from spheroids were more radio-resistant, with an absolute difference in mean survival fraction at 2 Gy of ~20%. The authors also examined several molecular assays relevant to radiosensitivity, including levels of reactive oxygen species (ROS) and phosphorylation of histone H2AX, both of which were decreased in spheroid cultures. Furthermore, fractionated radiation appeared to increase the percentage of nonadherent breast cancer stem cells, suggesting that the relative radioresistance of this subset may lead to their expansion during a course of radiotherapy. The relative resistance of normal mammary stem cells and progenitors to radiation was demonstrated recently in an article by Woodward and colleagues (27) . Progenitor cells in the mammary gland were more resistant to clinically relevant doses of radiation than nonprogenitor cells, which constituted the bulk of the mammary gland, and overexpression of the Wnt/β-catenin pathway could enhance the radioresistance of progenitor cells. Radiation also induced enrichment of SP progenitors (a marker of mammary stem cells) in the human breast cancer cell line MCF-7. Taken together, these data indicate that compared with differentiated cells, progenitor cells have different cell survival properties that may facilitate the development of targeted antiprogenitor cell therapies.


   INVOLVEMENT OF REACTIVE OXYGEN SPECIES IN CANCER PROMOTION AND PROGRESSION
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF CANCER STEM...
 CHEMORESISTANCE
 RADIORESISTANCE
 INVOLVEMENT OF REACTIVE OXYGEN...
CLINICAL IMPLICATIONS OF CANCER...
 REFERENCES
 
Reactive oxygen species such as superoxide (O2–) and its dismutation product hydrogen peroxide (H2O2) have been implicated in a host of disease states such as diabetes, cardiovascular and neurodegenerative disorders, inflammatory disease, mitochondrial pathologies, and cancer. At concentrations that do not overwhelm the cellular antioxidant defense systems, ROS such as O2– can function as proliferation signals via a direct or indirect effect on gene transcription and signal transduction circuitry (31 32 33) . Of note, ROS have been implicated in tumor initiation induced by a variety of agents in animal models of disease as well as in humans (34 35 36) . To that end, oxidative stress has been associated with the regulation of numerous cellular processes associated with the transformed phenotype, including DNA damage, proliferation, cell adhesion and migration, and cell survival or death signaling (34 , 37 38 39 40 41 42 43 44) . The critical determinant is the tight intracellular balance between superoxide and hydrogen peroxide levels, and a shift from the tightly regulated physiological ratio could affect cell fate decisions (45 46 47 48) . Similarly, a stimulatory effect of a mild increase in intracellular ROS on a variety of ion channels such as IP3 receptor-mediated Ca+2 mobilization, activities of Na+/Ca+2 and Na+/H+ (NHE) membrane transport pumps has been documented (31 , 49) . It is noteworthy that increased activity of these transport channels is observed during growth stimulation, and an alkaline intracellular milieu downstream of an active NHE exchanger activity promotes cell cycle progression/cell division and cell survival (38 , 50) . These cancer-promoting effects of O2– are corroborated by in vitro and in vivo experimental data demonstrating the tumor suppressor activity of the O2– scavenger MnSOD (51 52 53 54) . Furthermore, identification of the nonphagocytic mitogenic oxidase, Nox1, and its ability to stimulate growth via O2– further lends credence to the prooxidant theory of carcinogenesis (55) .

More convincing evidence linking a mild prooxidant state to cancer promotion/progression is provided by observations implicating O2– in oncogene-induced cellular transformation and/or cell survival. To that effect, a direct involvement of intracellular O2– in proliferation induced by the oncoprotein p21Ras was demonstrated in a lung fibroblast model via Ras-dependent activation of the GTPase Rac1 (56) . Cancer cells constitutively expressing the activated form of Rac1 (RasV12) maintained significantly elevated levels of O2– that correlated with resistance to drug-induced apoptosis, which was reversed upon expression of RacN17, a dominant-negative form of Rac1 (41) . The inhibitory effect of a slight prooxidant intracellular milieu on cell death signaling is further corroborated in a model of Bcl-2-mediated death inhibition. Bcl-2 is the first in a family of genes (classified as the Bcl-2 family) consisting of pro- and antiapoptotic members that serve as checkpoints or inducers of apoptosis signal transduction (57) . Historically, Bcl-2 has been described as an antioxidant due to its ability to suppress H2O2-induced lipid peroxidation (58) . However, recent data have challenged this notion by demonstrating that Bcl-2 does not elicit a direct antioxidant effect, but instead the slight prooxidant intracellular milieu on overexpression of Bcl-2 reinforces the cellular antioxidant defense systems. Along similar lines, the death inhibitory activity of Bcl-2 was linked to its ability to generate a slightly prooxidant intracellular environment in human leukemia cells (39) . The prooxidant activity of Bcl-2 appeared specific for intracellular O2– as evidenced by the ability of NADPH oxidase inhibitor, diphenyleneiodonium (DPI), or the dominant negative form of Rac1 to decrease O2– as well as enhanced apoptosis sensitivity in Bcl-2 overexpressing cells. These reports provide strong evidence linking oncogene-mediated cell survival and proliferation to a prooxidant intracellular milieu, and suggest an association between prooxidant state and tumor promotion/progression.

Thus, a permissive apoptotic milieu is a function of decreased intracellular O2– concentration and cytosolic acidification (47) . This has resulted in a paradigm shift in that ROS are no longer viewed as only being deleterious to cells and tissues. It is important to note that a prolife role for ROS is observed only under a mild prooxidant state, as the effects could be completely reversed if the concentration of ROS in the cells is high enough to trigger death signaling. Nothing is known on how the ROS status relates to cancer stem cells and their inherent/acquired ability to self-renew and/or evade death signals and thereby gain a definite survival advantage over their normal counterparts. Given the plethora of recent reports, it is plausible that a prooxidant intracellular milieu could be an acquired cancer trait to facilitate survival by fueling proliferative capacity at the expense of death signaling. Thus, manipulation of a cellular redox state might potentially serve as a strategy for modulating apoptosis in tumor cells and increasing their sensitivity to chemotherapeutic drugs.

Work by Smith et al. has uncovered the intracellular redox state modulation as a critical biochemical/molecular regulator of the balance between self-renewal and differentiation (59) . Modulation of the balance between self-renewing divisions and differentiation is at the heart of precursor cell function in development, tissue repair, and tissue homeostasis, and little is known about the physiological mechanisms central to such modulation. The work of Smith et al. demonstrated that the redox state could be modulated by cell-extrinsic molecules that altered the balance between self-renewal and differentiation: growth factors that promoted self-renewal caused progenitors to become more reduced while signaling molecules that promoted differentiation caused progenitors to become more oxidized. On the other hand, it has been suggested that the CD133+ cancer stem cell fraction in malignant brain tumors is correlated with enhanced chemoresistance and is increased in recurrent GBM tissue specimens (23) . Thus, by inference, it would suggest that the CD133+ cancer stem cell population in malignant brain tumors might possess an altered ROS regulatory mechanism that allowed them to maintain a state of self-renewal in response to insults by chemotherapeutic agents. This area remains to be elucidated.


   CLINICAL IMPLICATIONS OF CANCER STEM CELLS
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF CANCER STEM...
 CHEMORESISTANCE
 RADIORESISTANCE
 INVOLVEMENT OF REACTIVE OXYGEN...
 CLINICAL IMPLICATIONS OF CANCER...
REFERENCES
 
Despite the recent surge of published work in CSCs, the clinical significance of this population remains unclear. One prediction of the CSC model is that clinical behavior should depend largely on the CSC population, either in quantitative terms such as the relative or absolute number of CSCs or qualitative aspects related to biological features of CSCs. So far there are few data addressing this question, although a recent study suggests that, in AML, a higher percentage of blasts with the CD34+CD38– LSC phenotype is correlated with poorer overall survival (60) . Similar studies by Bao et al. in radioresistance in malignant brain tumors (GBM) showed that the percentage of CD133-expressing cells as analyzed by FACS also correlated with the rate of tumor formation when implanted in immunodeficient mice (25) .

Two recent studies by Bao et al. and Piccirillo et al. describe the implications of considering the functional hierarchy of the heterogeneous population of tumor cells and the subsequent identification of potential therapeutic targets for eradication of malignant brain tumors (25 , 61) . The implication of work by Bao and colleagues is that radiation treatment fails in the long run because it cannot kill the subpopulation of CD133+ tumor-initiating cells. These cells were rendered less resistant to radiation when two pharmacologic inhibitors of Chk1 and Chk2 kinases were added to disrupt the efficient DNA repair mechanisms of CD133+ cells. However, further work needs to be carried out on whether these treated cells also lost the ability to subsequently initiate tumors in vivo.

In other research, Piccirillo et al. first showed that human glioblastoma cells expressed bone morphogenetic proteins (BMPs) and their cell surface receptors, BMPs being the soluble factors that normally induce neural precursor cells to differentiate into mature astrocytes, a subtype of brain cells called glial cells (61) . The authors showed that BMPs could also prompt the differentiation of CD133+ brain tumor stem cells, critically weakening their tumor-forming ability. The results further imply that tumor populations at least partially retain a developmental hierarchy based on stem cells, and remain able to respond to the normal signals that induce them to mature. These findings should lead to renewed interest in devising therapies that promote the differentiation of cancer cells.

While the CSC hypothesis has exciting clinical implications, its widespread acceptance and application to the practice of medicine have yet to occur. Indeed, work by Liu and colleagues highlights that, in breast cancer, an "invasiveness" gene signature (IGS) that was differentially expressed in breast cancer stem cells compared with normal breast epithelium existed that differed significantly from gene signatures reported in breast cancer (62) . An important finding was that the IGS was associated with the risk of death and metastasis not only in breast cancer, but also in lung cancer, prostate cancer, and medulloblastoma (a pediatric brain tumor). The contribution of the breast cancer stem cell component of the IGS was essential to its association with the clinical outcome. These data suggest the clinical relevance of the tumorigenic subclass of breast cancer cells, the cancer stem cells.

We anticipate that in the coming years, CSCs will be identified in additional tumor types, and knowledge of the detailed biology and clinical significance of this experimentally defined population will provide further support for the CSC hypothesis. Ultimately, focusing research efforts on the CSC may drive important advances in our understanding of cancer biology and developing potential cures for these devastating diseases (Fig. 4 ).


Figure 4
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  		Figure 4. Therapeutic strategies. Current therapeutic strategies include targeting candidate cancer stem cells and their microenvironmental niche, which contributes to self-renewal of these cells. A potential novel strategy could involve targeting the ROS status of these cells, and tweaking their intracellular milieu to facilitate apoptotic death signals over proliferative effects.


   ACKNOWLEDGMENTS
 
C.T., B.T.A., and S.P. acknowledge funding support from the National Medical Research Council, and the Biomedical Research Council, Agency for Science, Technology and Research of Singapore. The authors thank Mr. Khaw Aik Kia for help with the art work.


   FOOTNOTES
 
1 C.T., National Neuroscience Institute, Dept of Research, 11 Jalan Tan Tock Seng, 308433 Singapore. E-mail: carolt{at}pacific.net.sg; S.P., Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, 117597 Singapore. E-mail: phssp{at}nus.edu.sg


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 IDENTIFICATION OF CANCER STEM...
 CHEMORESISTANCE
 RADIORESISTANCE
 INVOLVEMENT OF REACTIVE OXYGEN...
 CLINICAL IMPLICATIONS OF CANCER...
 REFERENCES
 

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