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REVIEW

Cancer stem cells: a new framework for the design of tumor therapies

Boyan K. Garvalov & Till Acker

Received: 14 July 2010 /Revised: 27 August 2010 /Accepted: 16 September 2010 # Springer-Verlag 2010

Abstract Modern tumor therapy has achieved considerable progress, but many tumors remain refractory to treatment or relapse following initial remission. Recent evidence points to one possible reason for this limited therapeutic efficiency: that the design of anticancer agents so far may not have been aimed at the right target. While conventional tumor therapies have targeted the main mass of tumor cells, there is now compelling evidence that tumor initiation and progression are driven by a subpopulation of tumor cells that possess stem cell properties and are resistant to traditional cancer treatments— the cancer stem cells (CSCs). CSCs have been identified in most types of cancer and can be separated from the rest of the tumor cells using appropriate markers. CSCs are regulated by molecular mechanisms and specific, perivascular, and hypox- ic microenvironments, which largely overlap with those controlling stem cells from normal tissues. Our improved understanding of CSC biology has already provided a number of novel targets and drug discovery platforms for the design of specific therapies that aim to eradicate the CSC subpopula- tion. Therapeutic approaches can be targeted either at eliminating the CSCs themselves or at disrupting the niches in which CSCs reside. Moreover, the importance of CSCs for tumor growth, resistance, and progression implies that clinical trials and preclinical studies of anticancer therapies should

include as a key element an assessment of the abundance and persistence of CSCs. Thus, CSC research holds great promise for providing important new impetus to the fields of tumor biology and clinical oncology.

Keywords Cancer stem cell . Hypoxia .

Microenvironment . Angiogenesis . Antitumor therapy.

Metastasis

The hierarchy model and cancer stem cells (CSCs)

The classical view of tumor formation is based on the “stochastic” or “clonal evolution” model [1, 2]. It perceives the tumor as a mass of hyperproliferative cells with similar potential for driving tumor growth. Tumor heterogeneity and progression are seen as the result of variations in the tumor microenvironment and genetic mutations in individ- ual cells, followed by selection of those that are best adapted to support the further growth of the tumor (Fig. 1a). An alternative concept that has been gaining increasing experimental support is the “hierarchy” or “cancer stem cell” model [3]. This model posits that tumors are generated and maintained in a manner similar to the physiological stem cell system operating in normal tissues, i.e., by cells with stem cell-like properties, which self-renew and differentiate into the distinct cellular subtypes of the tumor (Fig. 1b). The key novel features of this model are that only a limited population of tumor cells can drive tumor initiation and maintenance and that the heterogeneity of tumor cells is the result of differentiation from a stem cell precursor that sits on top of the tumor’s “differentiation hierarchy” [4], which may also include some level of plasticity (Fig. 1b). The CSC model, therefore, extends the clonal evolution model by proposing that clonal selection

B. K. Garvalov : T. Acker Institute of Neuropathology, Justus Liebig University, Giessen, Germany

T. Acker (*) Institute of Neuropathology, University Hospital Giessen and Marburg/Justus Liebig University, Arndtstr. 16, 35392 Giessen, Germany e-mail: [email protected]

J Mol Med DOI 10.1007/s00109-010-0685-3

operates at the level of CSCs. The notion that tumors may be initiated and maintained by aberrant cells with a stem cell character can be traced back to the nineteenth century and has been revived repeatedly ever since [5–7]. However, direct experimental evidence was first provided in the mid 1990s, when it was shown that acute myeloid leukemia could be transferred from human patients to immunodefi- cient mice by a rare population of cells, which carry markers of normal hematopoietic stem cells and reproduce the hierarchy of differentiated leukemic cells [8, 9]. These studies also set the standard by which CSCs are defined and identified: as those tumor cells that can self-renew and are capable of re-growing and reconstituting the heterogeneity of the tumor from which they were isolated.

Based on analogous experimental paradigms, CSCs have also been identified as a common feature of multiple solid tumors including lung [10, 11], colon [12–15], stomach [16], liver [17, 18], breast [19–21], prostate [22], pancreatic [23, 24], head and neck [25], skin [26], ovarian [27–29], bladder [30], mesenchymal cancer [31], brain tumors [32– 34], and melanoma [35–37] (Table 1). However, it has also become evident that there could be differences between tumor types, with some tumors containing a high propor- tion of tumorigenic cells and little CSC-dependent hierar- chy [38–41]. A variety of approaches are employed for the isolation of CSCs, often based on markers characteristic of normal stem cells, e.g., CD44, CD133, CD15, or ABC transporter proteins. Some of these markers may not be fully specific for CSCs, but in all cases, they provide a means to substantially enrich CSCs, which are more tumorigenic than the rest of the tumor cells. Importantly, CSC markers and signature genes have proven to be predictive of cancer progression and clinical outcome [21, 30, 37, 42–47]. Furthermore, more aggressive or refractory cancers contain more CSCs [37, 48], underscoring the clinical importance of this tumor cell population.

Properties and regulation of CSCs

The realization that CSCs represent a critical population in many tumors has prompted efforts to understand their properties in order to therapeutically target them more efficiently. CSCs share a number of features with normal stem cells. First of all, both CSCs and nonmalignant stem cells are defined by their ability to self-renew and differentiate. In vitro, CSCs are maintained in media supplemented with growth factors, but without serum [49]. Culturing under such conditions can also be used as a means of enriching the CSC population [15, 49, 50]. In such cultures, the self-renewal capacity of CSCs is typically measured by their ability to form three-dimensional spheres [51], similarly to normal neural or mammary stem cells [52, 53]. We have recently shown that the gene expression signature of CSCs reveals substantial overlaps with genes that are important for the maintenance of physiological stem cells [47]. Indeed signaling mechanisms regulating self-renewal and differentiation appear to be largely shared between CSCs and stem cells (Fig. 2). The Notch pathway, which plays an essential role in stem cell maintenance and differentiation throughout development, is important for CSC function. Activation of Notch signaling in glioma cells increases the expression of stem cell markers and enhances self-renewal [54, 55]. CSCs in medulloblastoma express high levels of Notch and are sensitive to inhibitors of the Notch pathway [56]. Similarly, Wnt signaling has a central function in the control of multi-/pluripotency, proliferation and differentiation in embryonic and adult stem cells and is also necessary for CSC self-renewal and tumorigenicity. The activation of the key Wnt downstream effector β- catenin (Fig. 2) is enhanced in skin CSCs and, strikingly, conditional ablation of the β-catenin gene in mouse skin cancer models led to a loss of CSCs and complete tumor regression [26]. Another example is the Hedgehog pathway,

Fig. 1 The stochastic and hierarchy (cancer stem cell) models of tumor development. a According to the stochastic model, tumor cells have similar proliferative capacity. Genetic mutation and selection of mutant clones (clonal evolution) confer different tumorigenicity to tumor cell subpopulations. b In the cancer stem cell model, only CSC can self-renew and give rise to tumor progenitor cells, which in turn

give rise to more differentiated tumor cells. The resulting tumor contains a hierarchy of cells, arising through the differentiation of stem-like cells. The CSCs, but not the differentiated tumor cells, can reinitiate tumor growth. However, the hierarchy may involve a certain degree of plasticity with more differentiated cells converting into less differentiated ones (dotted arrows)

J Mol Med

which is among the key regulators of stem cells from various tissues and also plays a critical role in CSCs. Inhibition of Hedgehog signaling suppressed CSC prolifer- ation and self-renewal and increased apoptosis [57]. Importantly, it was reported that glioblastoma cells remain viable after hedgehog blockade but are no longer capable of tumor initiation, indicating that the hedgehog pathway is required for maintaining the CSC population [58].

CSC niches

An additional similarity between CSCs and non-transformed stem cells is their dependence on particular niches— specialized microenvironments in which the surrounding

cells, extracellular matrix, and soluble factors are critical for the maintenance of cell stemness. In situ, CSCs have been found enriched in perivascular regions [59], and similar findings have been reported for neural stem cells [60–62]. Recent studies have started to shed light on the signaling mechanisms regulating CSCs in the perivascular niche. Medulloblastoma CSCs localized in the vicinity of blood vessels were resistant to radiation, owing to their ability to activate the PI3K/Akt pathway and undergo a transient, p53- dependent cell cycle arrest [63]. Intriguingly, the tumor vasculature in gliomas expresses high levels of endothelial nitric oxide synthase (eNOS). NO secreted from endothelial cells can activate a cGMP/PKG dependent pathway leading to enhanced Notch activity. This in turn leads to an increase of the CSC pool and its self-renewal capacity [64].

Table 1 Markers used for the identification and isolation of cancer stem cells from different cancers

Cancer type CSC markers % of CSC cells in tumor Efficiency of tumor formationa

(transplanted cells) Reference

Lung Sca-1+CD45–PECAM–CD34+ 0.008–0.064 ND [11]

CD133+ 0.3–22.0 ND [10]

Colon CD133 1.8–24.5 83% (500) [14, 15]

EpCAMhighCD44+ 0.03–38.0 (mean 5.4) 75% (200) [12]

ALDH1 3.5±1.0 ND (25) [13]

Breast CD44+EpCAM+CD24–Lineage– 0.6–5.0 100% (1,000) [19]

CD44+CD24-ALDH1+ 0.1–1.2 100% (20) [21]

Thy-1+CD24+CD45- 1.0–4.0 80% (50) [20]

Prostate CD44+α2β1 highCD133+ 0.1–0.3 ND [22]

Brain CD133 19–29 100% (100) [33]

CD15 5.6–70.5 100% (1,000) [34]

SP 0.15–1.2 ND [32]

Melanoma ABCB5 1.6–20.4 50% (100,000) [37]

Pancreatic CD44+CD24+EpCAM+ 0.2–0.8 50% (100) [24]

CD133 0.7–3.2 80% (500) [23]

Liver CD90+CD45– 0.7–6.2 50% (5000) [18]

Head and neck CD44 0.1–41.7 50% (5,000) [25]

Skin CD34 13.0–20.0 50% (1,000) [26]

Ovarian CD44+CD117+ 0.1–0.2 100% (100) [29]

CD133+ 0.3–35.0 (median 8.9) 83% (500) [28]

Bladder CD44 3.1–36.3 50% (1,000–3,000) [30]

Mesenchymal SP 0.07–10.5b 55% (100) [31]

Acute myeloid leukemia (AML) CD34++CD38– 0,02–2,00c 100%d (100,000–500,000) [8, 9]

Acute lymphoblastic leukemia (ALL)

CD34+CD10–/CD34+CD19– 8±4/3±1c 100%d (50,000–200,000) [116]

a The efficiency represents the percentage of immunodeficient mice that form tumors following injection of the number of cells indicated in brackets b Data presented only for a subset of tumors c Percentage among mononuclear cells d Percentage of engraftment

EpCAM epithelial cell adhesion molecule (also called epithelial-specific antigen), Lineage lineage markers: CD2, CD3, CD10, CD16, CD18, CD31, CD64, and CD140b, SP side population

J Mol Med

Moreover, genetic ablation of eNOS interfered with Notch activity and repressed tumor formation [64].

Importantly, the perinecrotic hypoxic micronenviron- ment was identified as a second niche where CSCs are concentrated, as we have recently demonstrated [47] again in analogy to normal stem cells [65]. Low oxygen levels (hypoxia) drive tumor progression by triggering a set of adaptive transcriptional responses that regulate tumor angiogenesis, metabolism, motility, and survival [66]. These cellular responses are primarily controlled by the transcription factor system of the hypoxia-inducible factors (HIFs). Interestingly, several reports have identified critical stem cell regulators such as Oct4 [67], c-myc [68], and Notch [69] as direct or indirect HIF targets. Importantly, several studies by us and others have directly demonstrated

that the CSC phenotype is also promoted by hypoxia and that the HIFs provide an intriguing link between the well- established function of hypoxia in tumor growth and stem cell biology. Hypoxia enhances the self-renewal of CSCs [47, 70, 71], while HIF knockdown blocks this effect and reduces CSC-mediated tumor growth [47, 72–74]. Interest- ingly, HIF-2α, but not HIF-1α, is highly expressed and strongly upregulated by hypoxia in glioma CSCs [72], enhances the expression of a set of CSC marker genes [47], and promotes tumor growth [70]. Taken together these findings underscore a critical role for the perivascular and the hypoxic niche in the maintenance and regulation of CSCs. The identity of the regulatory cellular components and molecular signals within these niches is one of the crucial open questions of CSC research.

Fig. 2 Principal pathways regulating CSCs. The scheme depicts the main components and clinically relevant targets involved in pathways that have been shown to control CSCs. The Notch pathway (orange) is activated by binding of transmembrane ligands such as Delta/Delta- like proteins or Jagged to the membrane receptor Notch. This induces cleavage of Notch by γ-secretase, releasing the Notch intracellular domain (NICD), which translocates to the nucleus and activates transcription in complex with cofactors of the CSL family. Different growth factors (e.g., EGF, FGF, PDGF) can activate receptor tyrosine kinases (RTKs, green) that subsequently promote the activity of phosphatidylinositol-3-kinase (PI3K), Akt and mammalian target of rapamycin (mTOR), among others, leading to enhanced protein translation, cell growth, and proliferation. Binding of Hedgehog (Hh, cyan) ligands to the receptor Patched (PTCH) relieves an inhibition of Smoothened (SMO), triggering a cascade that leads to the transloca- tion of glioma-associated oncogene homologue (Gli) into the nucleus and transcription of target genes. The Wnt pathway (yellow) is initiated by binding of Wnt ligands to a complex of the Frizzled (Fz)

and lipoprotein receptor-related protein (LRP) receptors. This sets off a series of signaling steps leading to stabilization of β-catenin, which can proceed to activate gene expression together with the TCF/LEF transcription factors. Endogenous Wnt inhibitors like Dickkopf proteins (DKK) and secreted Frizzled-related proteins (SFRPs) regulate the pathway and could be explored as tools for its pharmacological blockade. Specific factors in the CSC niches also play critical roles in regulating CSC self-renewal and differentiation. For example, NO produced by endothelial nitric oxide synthase (eNOS, red) can stimulate the production of cyclic guanosine monophosphate (cGMP) and activate protein kinase G (PKG), resulting in enhanced Notch signaling. Low oxygen tension in the hypoxic CSC niche suppresses the activity of prolyl hydroxylase domain-containing proteins (PHDs, blue), leading to stabilization of hypoxia-inducible factors (HIFs) and the transcription of HIF target genes. Notably, the HIF pathway interacts with other pathways, such as Notch [69], allowing for an intricate oxygen dependent crosstalk between CSC pathways

J Mol Med

CSCs have enhanced chemo-/radioresistance and metastatic potential

A common property of CSCs with major therapeutic significance is their enhanced resistance to standard anti- tumor treatments such as chemo- and radiotherapy [23, 63, 75–79]. Irradiation or chemotherapy in vivo enriches the fraction of cells expressing CSC markers, which also have an enhanced self-renewal capacity and tumorigenic- ity compared to the tumor bulk [23, 75, 77]. In addition, sorted CSC cells from different types of tumors survive such treatments in culture much better than unsorted or negative cells [75, 76, 78]. This is due to a combination of factors, including the high expression of ABC drug pumps, relative quiescence, resistance to oxidative DNA damage, and enhanced DNA repair capacity [37, 75]. The increased resistance of CSCs, combined with the ability of only a very small number of CSCs to reinitiate tumor growth [21, 33] is thought to be a major reason for cancer persistence and relapse after treatment. If CSCs are required for the initiation and growth of primary tumors, as posited in the hierarchy model, it is natural to assume that metastasis formation may also depend on CSCs. This is an issue of major clinical relevance since metastases are responsible for 90% of cancer related deaths, and indeed the involvement of CSCs in tumor dissemination has received experimental support in recent years. CSCs from mammary carcinoma have a phenotype similar to that of cells which have undergone epithelial–mesenchymal tran- sition, a process that plays a key role in tumor cell invasion and metastasis [80]. In addition, the early disseminating cells in the bone marrow of breast cancer patients have a CSC phenotype [81]. In pancreatic cancer CSCs are found at the invasive tumor front and determine the metastatic potential of the tumor [23]. Furthermore, a subpopulation of CD26+ colorectal CSCs possesses exclusive metastatic ability and is predictive of distant metastasis in colon cancer patients, in addition to exhibit- ing enhanced chemoresistance [82]. Taken together, these findings indicate that CSCs may be at the heart of the two key problems in tumor treatment—resistance to therapy and metastasis.

Consequences for cancer research and development of anticancer drug design platforms

The CSC paradigm has brought a fundamental shift in our understanding of cancer biology. It prompts a re- assessment of cancer as not simply a proliferative but also a differentiation disorder, in which tumor cells with aberrant stem cell characteristics that differentiate into abnormal progeny play a central role. From a technical

point of view, the discovery of CSCs has stimulated the development of better defined and more physiologically relevant experimental models. Our knowledge about CSCs questions the utility of long established cancer cell lines and culture systems, as well as standard subcutaneous tumor transplantations, as reliable tools for the study of tumor behavior. Established tumor cell lines have provided an easily tractable system to characterize many basic properties of CSCs, in particular when cultured under stem cell conditions [18, 47, 83, 84]. However, primary, low passage tumor cells isolated from tumor biopsies, continuously grown in non- differentiating conditions are both closer to the original tumors and maintain a subpopulation with characteristic CSC properties [49, 50, 85]. In addition, orthotopic, rather than subcutaneous tumor transplantation is the method of choice for assaying the defining property of CSCs, tumor propagation [19, 33].

The CSC model has a major impact on the focus of antitumor therapies, since it highlights the importance of eliminating the small but crucial and resilient subpopulation of CSCs (Fig. 3). In general, traditional strategies for drug design, which focus on the tumor bulk, have not proven to be suitable for the discovery of agents that eliminate CSCs. As discussed above, a hallmark of CSCs is the elevated resistance to classical chemo- and radiotherapy. Novel bulk tumor-targeted agents such as imatinib are also often ineffective against the CSC pool [86–88]. Conventional approaches for anticancer drug discovery target prolifer- ation, rather than self-renewal or differentiation. Standard end points include overall survival and proliferation in two-dimensional cell culture, as well as tumor size, metastasis and survival in vivo, predominantly in subcutaneous xenograft models. These parameters mostly reflect the behavior of the tumor bulk and are poor indicators of the drug’s efficacy against the CSC component. Therefore, to improve the clinical relevance of traditional drug screening analyses, it is important to complement them with an evaluation of treatment impact on CSCs. The influence of different agents on CSCs can be experimentally assessed using a number of established assays, such as analysis of CSC markers, signature genes, self-renewal assays (e.g. sphere formation [51]), and tumorigenicity assays in orthotopic xenotransplanta- tion models. Monitoring of the effect of anti-tumor treatments on the CSC pool should additionally be included as an important element in preclinical studies and clinical trials. CSCs can be assessed using biomarkers, e.g. surface markers (Table 1) or specific genetic signa- tures [47, 89]. The clinical relevance of CSCs for tumor growth and therapy responses underlines the necessity to develop new criteria and platforms for the design and assessment of CSC-targeted therapies.

J Mol Med

Novel, CSC-based drugs and drug targets

Our growing knowledge of the biology of CSCs and the application of improved drug discovery approaches, as discussed above, have provided a number of targets for the design of CSC depleting agents. In principle, two types of approaches targeting CSCs can be envisaged: eliminating the CSCs themselves by either killing or differentiating them, and disrupting the CSC niches (Fig. 3). One group of targets derives from pathways that are crucial in regulating CSC self-renewal mechanisms (see Fig. 2 and Table 2). For example, inhibition of Hedgehog signaling with cyclop- amine, an antagonist of the Hedgehog co-receptor SMO, killed CSCs in chronic myeloid leukemia and impaired the growth of imatinib resistant mouse and human CML cells [90]. A similar inhibitory effect of cyclopamine was observed in glioma, where it synergized with the standard chemotherapeutic temozolomide to suppress tumor growth [57]. Likewise, blockade of Notch signaling by inhibitors of γ-secretase, the protease required for Notch cleavage and activation, depleted CSCs in medulloblastoma and glioma, blocked tumor engraftment and growth and prolonged survival [56, 91]. Furthermore, administration of neutraliz- ing antibodies against the Notch ligand DLL4 reduced CSC frequency and tumor growth [92]. Wnt signaling may provide a further avenue for targeting CSCs, as it has been found to be required for CSC self-renewal and tumor growth in different cancers, including CML and squamous cell carcinoma [26, 93]. Additional signaling molecules, enriched or activated in CSCs, whose suppression inhibits tumor growth and increases survival in mouse models

include the NF-κB pathway [94, 95], IL-6 receptors [96], IL-3 receptor α (CD123) [97], integrin α6 [98], TGF-β, and LIF [99, 100]. The PI3K/Akt/PTEN/mTOR signaling axis (Fig. 2) is deregulated in many tumors, and in some settings, it can be used to preferentially target CSCs. CSCs have an increased sensitivity to Akt inhibition [101]. Additonally, inhibition of mTOR by rapamycin in a mouse model of PTEN-deficient leukemia depleted the CSC population and blocked the development of the disease [102]. In many of the above cases, the effect of the inhibitors may not depend on direct killing of CSCs but rather on promoting their differentiation into cells that no longer support tumor growth. For instance, skin tumor regression following ablation of Wnt signaling is accompanied by extensive terminal differentiation [26]. Similarly, activation of bone morphogenetic protein (BMP)-dependent signaling by BMP4 induced pronounced differentiation of glioblastoma cells, depleting the CSC pool and markedly attenuating tumorigenicity in a xenograft model [103]. Glioma CSC differentiation induced by retinoic acid sensitized the CSCs to therapy, impaired the secretion of angiogenic cytokines, inhibited motility, and reduced CSC tumorigenicity [104]. The identification of characteristic CSC surface markers (Table 1) provides further targets for the design of antibody- based antitumor agents (Table 2) that would either activate the body’s immune system against CSCs or would be coupled to cytotoxic agents [105]. In addition to candidate- based approaches for antitumor drug discovery, recent developments of suitable cell culture systems and in vitro assays have allowed high-throughput screens that produced promising leads against CSCs [85, 106].

Fig. 3 Comparison of conven- tional and CSC-based anticancer therapies. a Conventional thera- pies (brown flash) target the tumor bulk but are inefficient against CSCs (red), which can subsequently regrow the tumor. b CSC-based anticancer therapies are aimed at eliminating CSCs. One approach is either direct killing of CSCs or their differen- tiation into non-CSCs (orange flashes) that can be combinatori- ally targeted by standard treat- ments. Another strategy entails the disruption of CSC niches, such as hypoxic regions or peri- vascular regions, or niche-derived signals (blue flashes), which are required for CSC maintenance. Both approaches are additionally combined with conventional anti- cancer agents (brown flash) to destroy bulk tumor cells. Blood vessels are depicted in pink

J Mol Med

CSC niche targeting and combinatorial therapeutic approaches

Since CSCs carry the genetic mutations characteristic of the tumors they generate [107, 108], they may also exhibit a similar degree of genetic instability and mutation rate as the tumor bulk. Thus, therapies that exclusively target CSCs could run into the same problems that have overshadowed the design of anticancer drugs for decades: emergence of drug resistance and selection of increasingly refractory cell clones. One approach for circumventing these problems is presented by the dependence of CSCs on specific niches. Since the niches are primarily generated by non-mutated cells or cell-independent factors such as hypoxia, they may represent a more stable target for the elimination of CSCs. As discussed above, CSCs in solid tumors are enriched around blood vessels and depend on signals derived from them. Therefore, antiangiogenic therapies can be seen as a

promising strategy for depleting both the CSCs (by compromising their niche) and the tumor bulk (by depriving it from nutrients and oxygen). A number of antiangiogenic agents are approved or in clinical trials [109] (Table 2) and new targets are continuously being uncov- ered. For example, we have recently identified the VEGFR trafficking machinery as an additional target for inhibition of angiogenesis in tumors [110]. However, the antiangio- genic therapies that are currently available bring only limited benefits [111]. One explanation for this may be that by shrinking the tumor vasculature and blood supply, such agents stimulate the expansion of the other type of niche that has been described for CSCs—the hypoxic one [47]. This suggests that an additional important approach for targeting CSCs would involve the suppression of hypoxia-triggered signaling mechanisms. Substantial efforts are currently aimed at developing anticancer agents that target the HIF pathway [112]. Moreover, several known

Table 2 A selection of potential cancer stem cell inhibitors in (pre)clinical studies or approved for clinical use

Target/pathway Agent (Developer) Status Cancer indications References

Hedgehog

SMO antagonists GDC-0449 (Genentech/Curis) Phase I/II Solid tumors [117–119]

Cyclopamine Phase I Basal cell carcinoma [120]

Other IPI-926 (Infinity) Phase I Advanced/metastatic solid tumors [121]

BMS-833923/XL-139 (Bristol-Myers-Squibb/Exelixis)

Phase I Skin, solid tumors [122]

Notch

γ-Secretase inhibitors MK-0752 (Merck) Phase I Breast, pancreatic, brain cancer [123]

R4733 (Roche) Phase I/II Solid tumors [124]

PF-03084014 (Pfizer) Phase I Advanced cancer, leukemia [125]

DLL inhibitors anti-DLL4 mAbs Preclinical Solid tumors [126, 127]

Wnt

Inhibitors TCF/β-catenin inhibitors Preclinical Colon cancer [128–131]

Antibodies Wnt-2 mAb Preclinical Melanoma, non-small cell lung cancer [132, 133]

EpCAM

Antibodies Adecatumumab (Micromet) Phase II Metastatic breast cancer [134]

Edrecolomab Phase II-III Colon cancer [135, 136]

Catumaxomab (Trion/Fresenius) Approved Malignant ascites [137]

Angiogenesis

VEGF Bevacizumab (Genentech/Roche) Approved colon, non-small-cell lung cancer, breast cancer [138]

VEGF receptors Sorafenib (Bayer) Approved Renal carcinoma, hepatocellular carcinoma [139]

Sunitinib (Pfizer) Approved Renal carcinoma, gastrointestinal stromal tumors [140]

Hypoxia/HIF

PHD activators KRH102053 Preclinical Solid tumors [141]

HIF synthesis/activity PX-478 (Oncothyreon) Phase I Advanced solid tumors, lymphoma [142]

Topotecan (GlaxoSmithKline) Approved Ovarian, lung, cervical cancer [143]

Digoxin (GlaxoSmithKline) Approved Cardiac conditions [144]

Phase I-II Breast, lung cancer

FK228 (Gloucester) Phase II Solid tumors, lymphoma, [145]

17-AAG Phase I-II Solid tumors, leukemia [146]

J Mol Med

anticancer drugs that target regulators of HIF function have been shown to suppress HIF levels or activity [112] (Table 2). In principle, such agents could also play a two- fold role as both suppressors of bulk tumor growth and as inhibitors of CSCs, by targeting the hypoxic CSC niche. It should be noted, however, that most efforts so far have been focused on the more widely expressed and better studied member of the family, HIF-1α, whereas recent studies by us and others indicate that it is rather the second member of the family, HIF-2α, which plays a key role in the maintenance of CSCs [47, 72]. Future studies will need to address the activity of currently available agents targeting hypoxia signaling on CSCs, and to attempt designing specific HIF-2α inhibitors that block its function in CSC maintenance.

In designing future anticancer therapeutic strategies that include CSC eradication as a key element, it should be kept in mind that the approaches described above are unlikely to be successful when applied individually. In many cases, it may be essential to combine CSC-specific agents with traditional bulk tumor-targeting treatments (see Fig. 3). CSC depletion alone will be expected to halt tumor growth but will not necessarily reduce the size of preexisting tumors, which could impose a substantial clinical burden on the patient and should also be targeted, using traditional strategies. Similarly, drugs that cause CSC differentiation rather than death will require the elimination of the differentiated tumor cells as an additional step. The efficient eradication of CSCs themselves may require the combined ablation of CSCs and their niches. Furthermore, approaches that center on the CSC niche as a target will need to take into account the existence of different, even opposing (perivascular and hypoxic) niches and target them simulta- neously. Lastly, it has to be kept in mind that CSC targeting treatments may also deplete normal stem cells due to the molecular and functional similarities between CSCs and stem cells. However, recent studies have highlighted crucial differences between CSCs and stem cells that could be exploited for CSC specific treatments [72].

Conclusion and perspective

Based on our current knowledge, CSCs are likely to be a key driving force of most types of cancer. It is, therefore, justified to include the evaluation of CSC abundance and persistence as a critical element in future preclinical studies and clinical trials of antitumor therapies. The CSC markers that have been identified so far will greatly facilitate such analyses, but it is important to further improve our knowledge of additional markers with optimal specificity and sensitivity. The assessment of CSCs in solid tumors will be further aided if blood CSC biomarkers for solid

tumors become available, obviating the need for invasive tumor biopsies. Basic research into the properties of CSCs needs to further elucidate the mechanisms regulating the origin and maintenance of CSCs and to delineate differ- ences between CSCs and normal stem cells that could be exploited for specific CSC targeting with minimal side effects. The inclusion of CSC analyses in clinical studies of anticancer treatments will aid our understanding about the extent to which conclusions obtained in murine or tissue culture models, e.g. related to the radio- and chemo- resistance of CSCs, can be transferred to human patients. The functional assays used to assess CSCs should also be better standardized for specific tumor types. Additionally, some general issues in the CSC field remain to be clarified, related for instance to the origin of CSCs, their proliferative capacity, their abundance, their precise molecular charac- terization, or the extent to which differentiation and dedifferentiation occur within the tumor cell hierarchy [105, 113–115]. Overall, CSCs are likely to have a more variable and unstable character than physiological stem cells, just like tumors are more variable and unstable than healthy tissues. Furthermore, the differentiation hierarchy may not necessarily be unidirectional, as suggested by a recent in vitro study on melanoma showing that cells with CSC properties could be generated not only by self renewal but also by dedifferentiation from more differentiated progeny [79]. This would have direct therapeutic con- sequences, as CSC-based therapies would have to be administered chronically rather than as fast acting magic bullets to eradicate CSCs. It is also clear that the discovery of efficient drugs against CSCs will require specially tailored assays and screening platforms that rest upon our improved understanding of CSC characteristics. The com- bination of all these approaches has already started to yield a number of promising anticancer drug candidates. This new line of research holds great promise for significant improvements in the treatment of cancer by focusing on a novel and crucial type of target—the cancer stem cells.

Acknowledgments This work was supported by grants from the DFG (AC 110/3-1,-2 (SPP1190)), LOEWE (OSF), the Deutsche Krebshilfe (107231), the German Ministry of Education and Research (BMBF) within the National Genome Network (NGFNplus) and Brain Tumor Network, and VFK Krebsforschung.

Disclosure of potential conflict of interests The authors declare no conflict of interests related to this study.

References

1. Dalerba P, Cho RW, Clarke MF (2007) Cancer stem cells: models and concepts. Annu Rev Med 58:267–284

2. Nowell PC (1976) The clonal evolution of tumor cell popula- tions. Science 194:23–28

J Mol Med

3. Visvader JE, Lindeman GJ (2008) Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 8:755–768

4. Chen R, Nishimura MC, Bumbaca SM, Kharbanda S, Forrest WF, Kasman IM, Greve JM, Soriano RH, Gilmour LL, Rivers CS et al (2010) A hierarchy of self-renewing tumor-initiating cell types in glioblastoma. Cancer Cell 17:362–375

5. Bruce WR, Van Der Gaag H (1963) A quantitative assay for the number of murine lymphoma cells capable of proliferation in vivo. Nature 199:79–80

6. Hamburger AW, Salmon SE (1977) Primary bioassay of human tumor stem cells. Science 197:461–463

7. Wicha MS, Liu S, Dontu G (2006) Cancer stem cells: an old idea–a paradigm shift. Cancer Res 66:1883–1890, discussion 1895–1886

8. Bonnet D, Dick JE (1997) Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med 3:730–737

9. Lapidot T, Sirard C, Vormoor J, Murdoch B, Hoang T, Caceres- Cortes J, Minden M, Paterson B, Caligiuri MA, Dick JE (1994) A cell initiating human acute myeloid leukaemia after transplan- tation into SCID mice. Nature 367:645–648

10. Eramo A, Lotti F, Sette G, Pilozzi E, Biffoni M, Di Virgilio A, Conticello C, Ruco L, Peschle C, De Maria R (2008) Identification and expansion of the tumorigenic lung cancer stem cell population. Cell Death Differ 15:504–514

11. Kim CF, Jackson EL, Woolfenden AE, Lawrence S, Babar I, Vogel S, Crowley D, Bronson RT, Jacks T (2005) Identification of bronchioalveolar stem cells in normal lung and lung cancer. Cell 121:823–835

12. Dalerba P, Dylla SJ, Park IK, Liu R, Wang X, Cho RW, Hoey T, Gurney A, Huang EH, Simeone DM et al (2007) Phenotypic characterization of human colorectal cancer stem cells. Proc Natl Acad Sci USA 104:10158–10163

13. Huang EH, Hynes MJ, Zhang T, Ginestier C, Dontu G, Appelman H, Fields JZ, Wicha MS, Boman BM (2009) Aldehyde dehydrogenase 1 is a marker for normal and malignant human colonic stem cells (SC) and tracks SC overpopulation during colon tumorigenesis. Cancer Res 69:3382–3389

14. O’Brien CA, Pollett A, Gallinger S, Dick JE (2007) A human colon cancer cell capable of initiating tumour growth in immunodeficient mice. Nature 445:106–110

15. Ricci-Vitiani L, Lombardi DG, Pilozzi E, Biffoni M, Todaro M, Peschle C, De Maria R (2007) Identification and expansion of human colon-cancer-initiating cells. Nature 445:111–115

16. Takaishi S, Okumura T, Tu S, Wang SS, Shibata W, Vigneshwaran R, Gordon SA, Shimada Y, Wang TC (2009) Identification of gastric cancer stem cells using the cell surface marker CD44. Stem Cells 27:1006–1020

17. Ma S, Chan KW, Hu L, Lee TK, Wo JY, Ng IO, Zheng BJ, Guan XY (2007) Identification and characterization of tumorigenic liver cancer stem/progenitor cells. Gastroenterol- ogy 132:2542–2556

18. Yang ZF, Ho DW, Ng MN, Lau CK, Yu WC, Ngai P, Chu PW, Lam CT, Poon RT, Fan ST (2008) Significance of CD90+ cancer stem cells in human liver cancer. Cancer Cell 13:153–166

19. Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF (2003) Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci USA 100:3983–3988

20. Cho RW, Wang X, Diehn M, Shedden K, Chen GY, Sherlock G, Gurney A, Lewicki J, Clarke MF (2008) Isolation and molecular characterization of cancer stem cells in MMTV-Wnt-1 murine breast tumors. Stem Cells 26:364–371

21. Ginestier C, Hur MH, Charafe-Jauffret E, Monville F, Dutcher J, Brown M, Jacquemier J, Viens P, Kleer CG, Liu S et al (2007)

ALDH1 is a marker of normal and malignant human mammary stem cells and a predictor of poor clinical outcome. Cell Stem Cell 1:555–567

22. Collins AT, Berry PA, Hyde C, Stower MJ, Maitland NJ (2005) Prospective identification of tumorigenic prostate cancer stem cells. Cancer Res 65:10946–10951

23. Hermann PC, Huber SL, Herrler T, Aicher A, Ellwart JW, Guba M, Bruns CJ, Heeschen C (2007) Distinct populations of cancer stem cells determine tumor growth and metastatic activity in human pancreatic cancer. Cell Stem Cell 1:313–323

24. Li C, Heidt DG, Dalerba P, Burant CF, Zhang L, Adsay V, Wicha M, Clarke MF, Simeone DM (2007) Identification of pancreatic cancer stem cells. Cancer Res 67:1030–1037

25. Prince ME, Sivanandan R, Kaczorowski A, Wolf GT, Kaplan MJ, Dalerba P, Weissman IL, Clarke MF, Ailles LE (2007) Identification of a subpopulation of cells with cancer stem cell properties in head and neck squamous cell carcinoma. Proc Natl Acad Sci USA 104:973–978

26. Malanchi I, Peinado H, Kassen D, Hussenet T, Metzger D, Chambon P, Huber M, Hohl D, Cano A, Birchmeier W et al (2008) Cutaneous cancer stem cell maintenance is dependent on beta-catenin signalling. Nature 452:650–653

27. Bapat SA, Mali AM, Koppikar CB, Kurrey NK (2005) Stem and progenitor-like cells contribute to the aggressive behavior of human epithelial ovarian cancer. Cancer Res 65:3025–3029

28. Curley MD, Therrien VA, Cummings CL, Sergent PA, Koulouris CR, Friel AM, Roberts DJ, Seiden MV, Scadden DT, Rueda BR et al (2009) CD133 expression defines a tumor initiating cell population in primary human ovarian cancer. Stem Cells 27:2875–2883

29. Zhang S, Balch C, Chan MW, Lai HC, Matei D, Schilder JM, Yan PS, Huang TH, Nephew KP (2008) Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer Res 68:4311–4320

30. Chan KS, Espinosa I, Chao M, Wong D, Ailles L, Diehn M, Gill H, Presti J Jr, Chang HY, van de Rijn M et al (2009) Identification, molecular characterization, clinical prognosis, and therapeutic targeting of human bladder tumor-initiating cells. Proc Natl Acad Sci USA 106:14016–14021

31. Wu C, Wei Q, Utomo V, Nadesan P, Whetstone H, Kandel R, Wunder JS, Alman BA (2007) Side population cells isolated from mesenchymal neoplasms have tumor initiating potential. Cancer Res 67:8216–8222

32. Harris MA, Yang H, Low BE, Mukherje J, Guha A, Bronson RT, Shultz LD, Israel MA, Yun K (2008) Cancer stem cells are enriched in the side population cells in a mouse model of glioma. Cancer Res 68:10051–10059

33. Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB (2004) Identification of human brain tumour initiating cells. Nature 432:396–401

34. Son MJ, Woolard K, Nam DH, Lee J, Fine HA (2009) SSEA-1 is an enrichment marker for tumor-initiating cells in human glioblastoma. Cell Stem Cell 4:440–452

35. Fang D, Nguyen TK, Leishear K, Finko R, Kulp AN, Hotz S, Van Belle PA, Xu X, Elder DE, Herlyn M (2005) A tumorigenic subpopulation with stem cell properties in melanomas. Cancer Res 65:9328–9337

36. Monzani E, Facchetti F, Galmozzi E, Corsini E, Benetti A, Cavazzin C, Gritti A, Piccinini A, Porro D, Santinami M et al (2007) Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential. Eur J Cancer 43:935– 946

37. Schatton T, Murphy GF, Frank NY, Yamaura K, Waaga-Gasser AM, Gasser M, Zhan Q, Jordan S, Duncan LM, Weishaupt C et al (2008) Identification of cells initiating human melanomas. Nature 451:345–349

J Mol Med

38. Kelly PN, Dakic A, Adams JM, Nutt SL, Strasser A (2007) Tumor growth need not be driven by rare cancer stem cells. Science 317:337

39. Kennedy JA, Barabe F, Poeppl AG, Wang JC, Dick JE (2007) Comment on “Tumor growth need not be driven by rare cancer stem cells”. Science 318:1722, author reply 1722

40. le Viseur C, Hotfilder M, Bomken S, Wilson K, Rottgers S, Schrauder A, Rosemann A, Irving J, Stam RW, Shultz LD et al (2008) In childhood acute lymphoblastic leukemia, blasts at different stages of immunophenotypic maturation have stem cell properties. Cancer Cell 14:47–58

41. Quintana E, Shackleton M, Sabel MS, Fullen DR, Johnson TM, Morrison SJ (2008) Efficient tumour formation by single human melanoma cells. Nature 456:593–598

42. Liu R, Wang X, Chen GY, Dalerba P, Gurney A, Hoey T, Sherlock G, Lewicki J, Shedden K, Clarke MF (2007) The prognostic role of a gene signature from tumorigenic breast- cancer cells. N Engl J Med 356:217–226

43. Zeppernick F, Ahmadi R, Campos B, Dictus C, Helmke BM, Becker N, Lichter P, Unterberg A, Radlwimmer B, Herold- Mende CC (2008) Stem cell marker CD133 affects clinical outcome in glioma patients. Clin Cancer Res 14:123–129

44. Jiang F, Qiu Q, Khanna A, Todd NW, Deepak J, Xing L, Wang H, Liu Z, Su Y, Stass SA et al (2009) Aldehyde dehydrogenase 1 is a tumor stem cell-associated marker in lung cancer. Mol Cancer Res 7:330–338

45. Artells R, Moreno I, Diaz T, Martinez F, Gel B, Navarro A, Ibeas R, Moreno J, Monzo M (2010) Tumour CD133 mRNA expression and clinical outcome in surgically resected colorectal cancer patients. Eur J Cancer 46:642–649

46. Beier D, Wischhusen J, Dietmaier W, Hau P, Proescholdt M, Brawanski A, Bogdahn U, Beier CP (2008) CD133 expression and cancer stem cells predict prognosis in high-grade oligoden- droglial tumors. Brain Pathol 18:370–377

47. Seidel S, Garvalov BK, Wirta V, von Stechow L, Schänzer A, Meletis K, Wolter M, Sommerlad D, Henze AT, Nister M et al (2010) A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2α. Brain 133:983–995

48. van Rhenen A, Feller N, Kelder A, Westra AH, Rombouts E, Zweegman S, van der Pol MA, Waisfisz Q, Ossenkoppele GJ, Schuurhuis GJ (2005) High stem cell frequency in acute myeloid leukemia at diagnosis predicts high minimal residual disease and poor survival. Clin Cancer Res 11:6520–6527

49. Lee J, Kotliarova S, Kotliarov Y, Li A, Su Q, Donin NM, Pastorino S, Purow BW, Christopher N, Zhang W et al (2006) Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines. Cancer Cell 9:391–403

50. Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, Coradini D, Pilotti S, Pierotti MA, Daidone MG (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65:5506–5511

51. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, Squire J, Dirks PB (2003) Identification of a cancer stem cell in human brain tumors. Cancer Res 63:5821–5828

52. Dontu G, Abdallah WM, Foley JM, Jackson KW, Clarke MF, Kawamura MJ, Wicha MS (2003) In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells. Genes Dev 17:1253–1270

53. Vescovi AL, Parati EA, Gritti A, Poulin P, Ferrario M, Wanke E, Frolichsthal-Schoeller P, Cova L, Arcellana-Panlilio M, Colombo A et al (1999) Isolation and cloning of multipotential stem cells from the embryonic human CNS and establishment of transplantable human neural stem cell lines by epigenetic stimulation. Exp Neurol 156:71–83

54. Shih AH, Holland EC (2006) Notch signaling enhances nestin expression in gliomas. Neoplasia 8:1072–1082

55. Zhang XP, Zheng G, Zou L, Liu HL, Hou LH, Zhou P, Yin DD, Zheng QJ, Liang L, Zhang SZ et al (2008) Notch activation promotes cell proliferation and the formation of neural stem cell- like colonies in human glioma cells. Mol Cell Biochem 307:101–108

56. Fan X, Matsui W, Khaki L, Stearns D, Chun J, Li YM, Eberhart CG (2006) Notch pathway inhibition depletes stem-like cells and blocks engraftment in embryonal brain tumors. Cancer Res 66:7445–7452

57. Clement V, Sanchez P, de Tribolet N, Radovanovic I, Ruiz i Altaba A (2007) HEDGEHOG-GLI1 signaling regulates human glioma growth, cancer stem cell self-renewal, and tumorigenicity. Curr Biol 17:165–172

58. Bar EE, Chaudhry A, Lin A, Fan X, Schreck K, Matsui W, Piccirillo S, Vescovi AL, DiMeco F, Olivi A et al (2007) Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma. Stem Cells 25:2524– 2533

59. Calabrese C, Poppleton H, Kocak M, Hogg TL, Fuller C, Hamner B, Oh EY, Gaber MW, Finklestein D, Allen M et al (2007) A perivascular niche for brain tumor stem cells. Cancer Cell 11:69–82

60. Mirzadeh Z, Merkle FT, Soriano-Navarro M, Garcia-Verdugo JM, Alvarez-Buylla A (2008) Neural stem cells confer unique pinwheel architecture to the ventricular surface in neurogenic regions of the adult brain. Cell Stem Cell 3:265–278

61. Shen Q, Wang Y, Kokovay E, Lin G, Chuang SM, Goderie SK, Roysam B, Temple S (2008) Adult SVZ stem cells lie in a vascular niche: a quantitative analysis of niche cell-cell inter- actions. Cell Stem Cell 3:289–300

62. Tavazoie M, Van der Veken L, Silva-Vargas V, Louissaint M, Colonna L, Zaidi B, Garcia-Verdugo JM, Doetsch F (2008) A specialized vascular niche for adult neural stem cells. Cell Stem Cell 3:279–288

63. Hambardzumyan D, Becher OJ, Rosenblum MK, Pandolfi PP, Manova-Todorova K, Holland EC (2008) PI3K pathway regu- lates survival of cancer stem cells residing in the perivascular niche following radiation in medulloblastoma in vivo. Genes Dev 22:436–448

64. Charles N, Ozawa T, Squatrito M, Bleau AM, Brennan CW, Hambardzumyan D, Holland EC (2010) Perivascular nitric oxide activates notch signaling and promotes stem-like character in PDGF-induced glioma cells. Cell Stem Cell 6:141–152

65. Parmar K, Mauch P, Vergilio JA, Sackstein R, Down JD (2007) Distribution of hematopoietic stem cells in the bone marrow according to regional hypoxia. Proc Natl Acad Sci USA 104:5431–5436

66. Bertout JA, Patel SA, Simon MC (2008) The impact of O2 availability on human cancer. Nat Rev Cancer 8:967–975

67. Covello KL, Kehler J, Yu H, Gordan JD, Arsham AM, Hu CJ, Labosky PA, Simon MC, Keith B (2006) HIF-2alpha regulates Oct-4: effects of hypoxia on stem cell function, embryonic development, and tumor growth. Genes Dev 20:557–570

68. Gordan JD, Bertout JA, Hu CJ, Diehl JA, Simon MC (2007) HIF-2alpha promotes hypoxic cell proliferation by enhancing c-myc transcriptional activity. Cancer Cell 11:335–347

69. Gustafsson MV, Zheng X, Pereira T, Gradin K, Jin S, Lundkvist J, Ruas JL, Poellinger L, Lendahl U, Bondesson M (2005) Hypoxia requires notch signaling to maintain the undifferentiated cell state. Dev Cell 9:617–628

70. Heddleston JM, Li Z, McLendon RE, Hjelmeland AB, Rich JN (2009) The hypoxic microenvironment maintains glioblastoma stem cells and promotes reprogramming towards a cancer stem cell phenotype. Cell Cycle 8:3274–3284

J Mol Med

71. McCord AM, Jamal M, Shankavaram UT, Lang FF, Camphausen K, Tofilon PJ (2009) Physiologic oxygen concentration enhances the stem-like properties of CD133+ human glioblastoma cells in vitro. Mol Cancer Res 7:489–497

72. Li Z, Bao S, Wu Q, Wang H, Eyler C, Sathornsumetee S, Shi Q, Cao Y, Lathia J, McLendon RE et al (2009) Hypoxia-inducible factors regulate tumorigenic capacity of glioma stem cells. Cancer Cell 15:501–513

73. Mendez O, Zavadil J, Esencay M, Lukyanov Y, Santovasi D, Wang SC, Newcomb EW, Zagzag D (2010) Knock down of HIF- 1alpha in glioma cells reduces migration in vitro and invasion in vivo and impairs their ability to form tumor spheres. Mol Cancer 9:133

74. Soeda A, Park M, Lee D, Mintz A, Androutsellis-Theotokis A, McKay RD, Engh J, Iwama T, Kunisada T, Kassam AB et al (2009) Hypoxia promotes expansion of the CD133-positive glioma stem cells through activation of HIF-1alpha. Oncogene 28:3949–3959

75. Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN (2006) Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature 444:756–760

76. Eramo A, Ricci-Vitiani L, Zeuner A, Pallini R, Lotti F, Sette G, Pilozzi E, Larocca LM, Peschle C, De Maria R (2006) Chemotherapy resistance of glioblastoma stem cells. Cell Death Differ 13:1238–1241

77. Li X, Lewis MT, Huang J, Gutierrez C, Osborne CK, Wu MF, Hilsenbeck SG, Pavlick A, Zhang X, Chamness GC et al (2008) Intrinsic resistance of tumorigenic breast cancer cells to chemotherapy. J Natl Cancer Inst 100:672–679

78. Ma S, Lee TK, Zheng BJ, Chan KW, Guan XY (2008) CD133+ HCC cancer stem cells confer chemoresistance by preferential expression of the Akt/PKB survival pathway. Oncogene 27:1749–1758

79. Roesch A, Fukunaga-Kalabis M, Schmidt EC, Zabierowski SE, Brafford PA, Vultur A, Basu D, Gimotty P, Vogt T, Herlyn M (2010) A temporarily distinct subpopulation of slow-cycling melanoma cells is required for continuous tumor growth. Cell 141:583–594

80. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M et al (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell 133:704–715

81. Balic M, Lin H, Young L, Hawes D, Giuliano A, McNamara G, Datar RH, Cote RJ (2006) Most early disseminated cancer cells detected in bone marrow of breast cancer patients have a putative breast cancer stem cell phenotype. Clin Cancer Res 12:5615– 5621

82. Pang R, Law WL, Chu ACY, Poon JT, Lam CSC, Chow AKM, Ng L, Cheung LWH, Lan XR, Lan HY et al (2010) A subpopulation of CD26+ cancer stem cells with metastatic capacity in human colorectal cancer. Cell Stem Cell 6:603– 615

83. Charafe-Jauffret E, Ginestier C, Iovino F, Wicinski J, Cervera N, Finetti P, Hur MH, Diebel ME, Monville F, Dutcher J et al (2009) Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res 69:1302–1313

84. Kondo T, Setoguchi T, Taga T (2004) Persistence of a small subpopulation of cancer stem-like cells in the C6 glioma cell line. Proc Natl Acad Sci USA 101:781–786

85. Pollard SM, Yoshikawa K, Clarke ID, Danovi D, Stricker S, Russell R, Bayani J, Head R, Lee M, Bernstein M et al (2009) Glioma stem cell lines expanded in adherent culture have tumor- specific phenotypes and are suitable for chemical and genetic screens. Cell Stem Cell 4:568–580

86. Bhatia R, Holtz M, Niu N, Gray R, Snyder DS, Sawyers CL, Arber DA, Slovak ML, Forman SJ (2003) Persistence of malignant hematopoietic progenitors in chronic myelogenous leukemia patients in complete cytogenetic remission following imatinib mesylate treatment. Blood 101:4701–4707

87. Copland M, Hamilton A, Elrick LJ, Baird JW, Allan EK, Jordanides N, Barow M, Mountford JC, Holyoake TL (2006) Dasatinib (BMS-354825) targets an earlier progenitor population than imatinib in primary CML but does not eliminate the quiescent fraction. Blood 107:4532–4539

88. Graham SM, Jorgensen HG, Allan E, Pearson C, Alcorn MJ, Richmond L, Holyoake TL (2002) Primitive, quiescent, Philadelphia-positive stem cells from patients with chronic myeloid leukemia are insensitive to STI571 in vitro. Blood 99:319–325

89. Günther HS, Schmidt NO, Phillips HS, Kemming D, Kharbanda S, Soriano R, Modrusan Z, Meissner H, Westphal M, Lamszus K (2008) Glioblastoma-derived stem cell-enriched cultures form distinct subgroups according to molecular and phenotypic criteria. Oncogene 27:2897–2909

90. Zhao C, Chen A, Jamieson CH, Fereshteh M, Abrahamsson A, Blum J, Kwon HY, Kim J, Chute JP, Rizzieri D et al (2009) Hedgehog signalling is essential for maintenance of cancer stem cells in myeloid leukaemia. Nature 458:776–779

91. Fan X, Khaki L, Zhu TS, Soules ME, Talsma CE, Gul N, Koh C, Zhang J, Li YM, Maciaczyk J et al (2010) NOTCH pathway blockade depletes CD133-positive glioblastoma cells and inhib- its growth of tumor neurospheres and xenografts. Stem Cells 28:5–16

92. Hoey T, Yen WC, Axelrod F, Basi J, Donigian L, Dylla S, Fitch- Bruhns M, Lazetic S, Park IK, Sato A et al (2009) DLL4 blockade inhibits tumor growth and reduces tumor-initiating cell frequency. Cell Stem Cell 5:168–177

93. Zhao C, Blum J, Chen A, Kwon HY, Jung SH, Cook JM, Lagoo A, Reya T (2007) Loss of beta-catenin impairs the renewal of normal and CML stem cells in vivo. Cancer Cell 12:528–541

94. Guzman ML, Swiderski CF, Howard DS, Grimes BA, Rossi RM, Szilvassy SJ, Jordan CT (2002) Preferential induction of apoptosis for primary human leukemic stem cells. Proc Natl Acad Sci USA 99:16220–16225

95. Hjelmeland AB, Wu Q, Wickman S, Eyler C, Heddleston J, Shi Q, Lathia JD, Macswords J, Lee J, McLendon RE et al (2010) Targeting A20 decreases glioma stem cell survival and tumor growth. PLoS Biol 8:e1000319

96. Wang H, Lathia JD, Wu Q, Wang J, Li Z, Heddleston JM, Eyler CE, Elderbroom J, Gallagher J, Schuschu J et al (2009) Targeting interleukin 6 signaling suppresses glioma stem cell survival and tumor growth. Stem Cells 27:2393–2404

97. Jin L, Lee EM, Ramshaw HS, Busfield SJ, Peoppl AG, Wilkinson L, Guthridge MA, Thomas D, Barry EF, Boyd A et al (2009) Monoclonal antibody-mediated targeting of CD123, IL-3 receptor alpha chain, eliminates human acute myeloid leukemic stem cells. Cell Stem Cell 5:31–42

98. Lathia JD, Gallagher J, Heddleston JM, Wang J, Eyler CE, Macswords J, Wu Q, Vasanji A, McLendon RE, Hjelmeland AB et al (2010) Integrin alpha 6 regulates glioblastoma stem cells. Cell Stem Cell 6:421–432

99. Ikushima H, Todo T, Ino Y, Takahashi M, Miyazawa K, Miyazono K (2009) Autocrine TGF-beta signaling maintains tumorigenicity of glioma-initiating cells through Sry-related HMG-box factors. Cell Stem Cell 5:504–514

100. Peñuelas S, Anido J, Prieto-Sanchez RM, Folch G, Barba I, Cuartas I, Garcia-Dorado D, Poca MA, Sahuquillo J, Baselga J et al (2009) TGF-beta increases glioma-initiating cell self-renewal through the induction of LIF in human glioblastoma. Cancer Cell 15:315–327

J Mol Med

101. Eyler CE, Foo WC, LaFiura KM, McLendon RE, Hjelmeland AB, Rich JN (2008) Brain cancer stem cells display preferential sensitivity to Akt inhibition. Stem Cells 26:3027–3036

102. Yilmaz OH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ (2006) Pten dependence distinguishes haemato- poietic stem cells from leukaemia-initiating cells. Nature 441:475–482

103. Piccirillo SG, Reynolds BA, Zanetti N, Lamorte G, Binda E, Broggi G, Brem H, Olivi A, Dimeco F, Vescovi AL (2006) Bone morphogenetic proteins inhibit the tumorigenic potential of human brain tumour-initiating cells. Nature 444:761–765

104. Campos B, Wan F, Farhadi M, Ernst A, Zeppernick F, Tagscherer KE, Ahmadi R, Lohr J, Dictus C, Gdynia G et al (2010) Differentiation therapy exerts antitumor effects on stem- like glioma cells. Clin Cancer Res 16:2715–2728

105. Zhou BB, Zhang H, Damelin M, Geles KG, Grindley JC, Dirks PB (2009) Tumour-initiating cells: challenges and opportunities for anticancer drug discovery. Nat Rev Drug Discov 8:806–823

106. Gupta PB, Onder TT, Jiang G, Tao K, Kuperwasser C, Weinberg RA, Lander ES (2009) Identification of selective inhibitors of cancer stem cells by high-throughput screening. Cell 138:645– 659

107. Eisterer W, Jiang X, Christ O, Glimm H, Lee KH, Pang E, Lambie K, Shaw G, Holyoake TL, Petzer AL et al (2005) Different subsets of primary chronic myeloid leukemia stem cells engraft immunodeficient mice and produce a model of the human disease. Leukemia 19:435–441

108. Magnifico A, Albano L, Campaner S, Delia D, Castiglioni F, Gasparini P, Sozzi G, Fontanella E, Menard S, Tagliabue E (2009) Tumor-initiating cells of HER2-positive carcinoma cell lines express the highest oncoprotein levels and are sensitive to trastuzumab. Clin Cancer Res 15:2010–2021

109. Cao Y (2009) Tumor angiogenesis and molecular targets for therapy. Front Biosci 14:3962–3973

110. Sawamiphak S, Seidel S, Essmann CL, Wilkinson GA, Pitulescu ME, Acker T, Acker-Palmer A (2010) Ephrin-B2 regulates VEGFR2 function in developmental and tumour angiogenesis. Nature 465:487–491

111. Bergers G, Hanahan D (2008) Modes of resistance to anti- angiogenic therapy. Nat Rev Cancer 8:592–603

112. Poon E, Harris AL, Ashcroft M (2009) Targeting the hypoxia- inducible factor (HIF) pathway in cancer. Expert Rev Mol Med 11:e26

113. Gupta PB, Chaffer CL, Weinberg RA (2009) Cancer stem cells: mirage or reality? Nat Med 15:1010–1012

114. Jordan CT (2009) Cancer stem cells: controversial or just misunderstood? Cell Stem Cell 4:203–205

115. Shackleton M, Quintana E, Fearon ER, Morrison SJ (2009) Heterogeneity in cancer: cancer stem cells versus clonal evolution. Cell 138:822–829

116. Cox CV, Evely RS, Oakhill A, Pamphilon DH, Goulden NJ, Blair A (2004) Characterization of acute lymphoblastic leukemia progenitor cells. Blood 104:2919–2925

117. Robarge KD, Brunton SA, Castanedo GM, Cui Y, Dina MS, Goldsmith R, Gould SE, Guichert O, Gunzner JL, Halladay J et al (2009) GDC-0449-a potent inhibitor of the hedgehog pathway. Bioorg Med Chem Lett 19:5576–5581

118. Rudin CM, Hann CL, Laterra J, Yauch RL, Callahan CA, Fu L, Holcomb T, Stinson J, Gould SE, Coleman B et al (2009) Treatment of medulloblastoma with hedgehog pathway inhibitor GDC-0449. N Engl J Med 361:1173–1178

119. Von Hoff DD, LoRusso PM, Rudin CM, Reddy JC, Yauch RL, Tibes R, Weiss GJ, Borad MJ, Hann CL, Brahmer JR et al (2009) Inhibition of the hedgehog pathway in advanced basal-cell carcinoma. N Engl J Med 361:1164–1172

120. Tabs S, Avci O (2004) Induction of the differentiation and apoptosis of tumor cells in vivo with efficiency and selectivity. Eur J Dermatol 14:96–102

121. Olive KP, Jacobetz MA, Davidson CJ, Gopinathan A, McIntyre D, Honess D, Madhu B, Goldgraben MA, Caldwell ME, Allard D et al (2009) Inhibition of hedgehog signaling enhances delivery of chemotherapy in a mouse model of pancreatic cancer. Science 324:1457–1461

122. Tremblay MR, Nesler M, Weatherhead R, Castro AC (2009) Recent patents for hedgehog pathway inhibitors for the treatment of malignancy. Expert Opin Ther Pat 19:1039–1056

123. Olson RE, Albright CF (2008) Recent progress in the medicinal chemistry of gamma-secretase inhibitors. Curr Top Med Chem 8:17–33

124. Winquist RJ, Boucher DM, Wood M, Furey BF (2009) Targeting cancer stem cells for more effective therapies: taking out cancer’s locomotive engine. Biochem Pharmacol 78:326–334

125. Wei P, Walls M, Qiu M, Ding R, Denlinger RH, Wong A, Tsaparikos K, Jani JP, Hosea N, Sands M et al (2010) Evaluation of selective gamma-secretase inhibitor PF-03084014 for its antitumor efficacy and gastrointestinal safety to guide optimal clinical trial design. Mol Cancer Ther 9:1618–1628

126. Noguera-Troise I, Daly C, Papadopoulos NJ, Coetzee S, Boland P, Gale NW, Lin HC, Yancopoulos GD, Thurston G (2006) Blockade of Dll4 inhibits tumour growth by promoting non- productive angiogenesis. Nature 444:1032–1037

127. Ridgway J, Zhang G, Wu Y, Stawicki S, Liang WC, Chanthery Y, Kowalski J, Watts RJ, Callahan C, Kasman I et al (2006) Inhibition of Dll4 signalling inhibits tumour growth by dereg- ulating angiogenesis. Nature 444:1083–1087

128. Chen Z, Venkatesan AM, Dehnhardt CM, Dos Santos O, Delos Santos E, Ayral-Kaloustian S, Chen L, Geng Y, Arndt KT, Lucas J et al (2009) 2, 4-Diamino-quinazolines as inhibitors of beta- catenin/Tcf-4 pathway: Potential treatment for colorectal cancer. Bioorg Med Chem Lett 19:4980–4983

129. Eguchi M, Nguyen C, Lee SC, Kahn M (2005) ICG-001, a novel small molecule regulator of TCF/beta-catenin transcription. Med Chem 1:467–472

130. Lepourcelet M, Chen YN, France DS, Wang H, Crews P, Petersen F, Bruseo C, Wood AW, Shivdasani RA (2004) Small- molecule antagonists of the oncogenic Tcf/beta-catenin protein complex. Cancer Cell 5:91–102

131. Takahashi-Yanaga F, Kahn M (2010) Targeting Wnt signaling: can we safely eradicate cancer stem cells? Clin Cancer Res 16:3153–3162

132. You L, He B, Xu Z, Uematsu K, Mazieres J, Fujii N, Mikami I, Reguart N, McIntosh JK, Kashani-Sabet M et al (2004) An anti- Wnt-2 monoclonal antibody induces apoptosis in malignant melanoma cells and inhibits tumor growth. Cancer Res 64:5385– 5389

133. You L, He B, Xu Z, Uematsu K, Mazieres J, Mikami I, Reguart N, Moody TW, Kitajewski J, McCormick F et al (2004) Inhibition of Wnt-2-mediated signaling induces programmed cell death in non-small-cell lung cancer cells. Oncogene 23:6170–6174

134. Naundorf S, Preithner S, Mayer P, Lippold S, Wolf A, Hanakam F, Fichtner I, Kufer P, Raum T, Riethmuller G et al (2002) In vitro and in vivo activity of MT201, a fully human monoclonal antibody for pancarcinoma treatment. Int J Cancer 100:101–110

135. Hartung G, Hofheinz RD, Dencausse Y, Sturm J, Kopp- Schneider A, Dietrich G, Fackler-Schwalbe I, Bornbusch D, Gonnermann M, Wojatschek C et al (2005) Adjuvant therapy with edrecolomab versus observation in stage II colon cancer: a multicenter randomized phase III study. Onkologie 28:347–350

136. Adams GP, Weiner LM (2005) Monoclonal antibody therapy of cancer. Nat Biotechnol 23:1147–1157

J Mol Med

137. Seimetz D, Lindhofer H, Bokemeyer C (2010) Development and approval of the trifunctional antibody catumaxomab (anti- EpCAMxanti-CD3) as a targeted cancer immunotherapy. Cancer Treat Rev 36(6):458–467

138. Willett CG, Boucher Y, di Tomaso E, Duda DG, Munn LL, Tong RT, Chung DC, Sahani DV, Kalva SP, Kozin SVet al (2004) Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 10:145–147

139. Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, Schwartz B, Simantov R, Kelley S (2006) Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 5:835–844

140. Faivre S, Demetri G, Sargent W, Raymond E (2007) Molecular basis for sunitinib efficacy and future clinical development. Nat Rev Drug Discov 6:734–745

141. Choi HJ, Song BJ, Gong YD, Gwak WJ, Soh Y (2008) Rapid degradation of hypoxia-inducible factor-1alpha by KRH102053, a new activator of prolyl hydroxylase 2. Br J Pharmacol 154:114–125

142. Welsh S, Williams R, Kirkpatrick L, Paine-Murrieta G, Powis G (2004) Antitumor activity and pharmacodynamic properties of

PX-478, an inhibitor of hypoxia-inducible factor-1alpha. Mol Cancer Ther 3:233–244

143. Rapisarda A, Zalek J, Hollingshead M, Braunschweig T, Uranchimeg B, Bonomi CA, Borgel SD, Carter JP, Hewitt SM, Shoemaker RH et al (2004) Schedule-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation, angiogen- esis, and tumor growth by topotecan in U251-HRE glioblastoma xenografts. Cancer Res 64:6845–6848

144. Zhang H, Qian DZ, Tan YS, Lee K, Gao P, Ren YR, Rey S, Hammers H, Chang D, Pili R et al (2008) Digoxin and other cardiac glycosides inhibit HIF-1alpha synthesis and block tumor growth. Proc Natl Acad Sci USA 105:19579–19586

145. Mie Lee Y, Kim SH, Kim HS, Jin Son M, Nakajima H, Jeong Kwon H, Kim KW (2003) Inhibition of hypoxia-induced angiogenesis by FK228, a specific histone deacetylase inhibitor, via suppression of HIF-1alpha activity. Biochem Biophys Res Commun 300:241–246

146. Kim WY, Oh SH, Woo JK, Hong WK, Lee HY (2009) Targeting heat shock protein 90 overrides the resistance of lung cancer cells by blocking radiation-induced stabilization of hypoxia- inducible factor-1alpha. Cancer Res 69:1624–1632

J Mol Med