Cancer Stem Cells: Potential Mediators of Therapeutic Resistance and Novel Targets of Anti-cancer Treatments. Hong Yan, Jichao Qin, and Dean G. Tang.
Cancer Stem Cells: Potential Mediators of Therapeutic Resistance and Novel Targets of Anti-cancer Treatments Hong Yan, Jichao Qin, and Dean G. Tang
1 Introduction Over the last decade, anti-cancer therapies (chemotherapy and radiation, hormonal, neoadjuvant, and combinatorial therapies) have prolonged the lives of cancer patients. However, present cancer therapies fail in a high percentage of cases due to an incomplete elimination of the tumor cells, resulting in relapse and metastasis of the tumor. The vast majority of cancer-related deaths are due to metastatic tumor growth that impairs the function of vital organ(s). Thus, cancer relapse and metastasis are the major challenges in fighting cancer. Metastasis is an inefficient process in that 100-fold enrichment
2500 CD133+ cells generate T 25 CD 133+ -derived spheres generate T 150 EpCAMCD 166+ CD44+ cells generate T
3000 CD133+ cells generate T
Positive cells are more clonogenic 1 CSC/57,000 T. cells 1 CSC/ 262 CD133+ cells
500 ALDH+ cells generate T; 20 ALDH+ CD44+ CD24 Lin cells CD133+ more tumorigenic
>50-fold enrichment
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19 (18 primary, 1, met)
30 primary (2 pre-cancerous)
Lung cancer
Liver cancer Ovarian cancer
4 wk SCID or nude mice SCID/Beige mice 3–4 wk nude mice
CD133+ (FACS)
CD44
+
CD117
+
(FACS)
CD133CD90+ (MACS)
Mice NOD/SCID mice
Marker
Table 1 (continued)
ABCB5+(MACS)
1. AI-Hajj M, et al. PNAS 2003; 100:3983–8. 2. Ginestier C et al. Cell Stom cell 2007; 1:555–67. 3. Singh SK, et al. Nature 2004; 432:396–401. 4. Collins AT, et al. Cancer Res 2005; 65:10946–51. 5. O’ Brien CA, et al. Cells Stem Cell 2007; 1:389–402. 6. Ricci-vitiani L, et al. Nature 2007; 445:111–5. 7. Todaro M et al. Cell Stem Cell 2007; 1–389–402. 8. Dalerba P et al. PNAS 2007; 104:10158–63. 9. Li C et al. Cancer Res. 2007; 67:1030–7. 10. Hemann PC et al. Cell 2007; 1:313–32. 11. Prince ME et al. PNAS 2007; 104:973–8. 12. Schatton T, et al. Nature 2008; 451:345–9. 13. Eramo A et al. Cell Death Differ, 2008; 15:504–14. 14. Yang ZF et al. Cancer Cell 2008; 13:153–66. 15. Zhang S et al. Cancer Res. 2008; 68:4311–20.
2 xenograft (from sphere) B primary (1 fresh 2 cultured to spheroid)
7 (1 primary; 4 LN, and 2 visceral met.)
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Tumor type Transplantation
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CD45 CD90+ more tumorigenic CD44+ CD117+ more tumorigenic
1 MMIC/1 million bulk T cells; 1st xeno: 1 MMIC/160,000 ABCB5+ cells, 2ary xeno: 1 MMIC/120,000 ABCB5+ cells 104 CD133+ cells generate T
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How should a CSC be defined? A CSC is a cell within a tumor that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor, and the regenerated tumors can be serially xenotransplanted [20, 24]. Strictly speaking, none of the CSCs thus far reported can be truly classified as CSCs and should, more appropriately, be called ‘‘tumorreinitiating cells,’’ ‘‘tumorigenic cells,’’ or ‘‘putative CSCs.’’ In reality, it will be very difficult to identify a tumorigenic cell that can fulfill the strict definition of CSCs mentioned above. First, a tumor, especially a solid tumor, is made of numerous cell types. To expect one cell or even a population of cells, when transplanted into a foreign host (i.e., mice) in an exotic environment, to fully reconstitute an original patient tumor in its complete composition begs tremendous imagination and will be essentially impossible to prove. Second, when such experiments are actually done, the best one can do is to co-inject the putative tumorigenic cell population with stromal cells (e.g., fibroblasts) in an extracellular matrix (e.g., matrigel or collagen gel) into an ‘‘orthotopic’’ animal site such as brain, mammary fat pad, or prostate lobes. These so-called orthotopic sites are considerably different from their human counterparts and tumor establishment would inevitably require the recruitment of various host (i.e., mouse) cells by the tumor-reinitiating cells. One can be certain that such ‘‘reconstituted’’ tumors could never be the same as the original patient tumors. Third, during the purification process, the majority of cells are often discarded to obtain marker-positive and marker-negative populations. These discarded cells would be important in the original tumor composition but they would be very difficult to reconstitute in tumor development assays. Altogether, one can say, at the best, that the experimental tumor reconstituted from the presumptive CSCs histologically ‘‘resembles’’ the patient tumor [24].
2.2 Identification 2.2.1 Marker-Based Analysis This is the most widely used and also the most practical approach. A variety of adult tissue SCs are found to express relatively specific markers, which can be cell surface or intracellular (e.g., nuclear and cytoskeletal). Interestingly, CSCs seem to express various cell surface markers such as CD44 and/or CD133 that identify their normal counterparts [25], which allows a relatively simple enrichment procedure by utilizing either flow cytometry-based cell sorting or microbeadsbased affinity purification. For intracellular markers such as a nuclear or cytoskeletal protein, a gene promoter-driven reporter construct such as GFPtagged retroviral or lentiviral vector system can be developed to track down and purify putative (cancer) SCs [26]. Alternatively, transgenic animal models can be made by knocking in the gene promoter-driven reporter (GFP, LacZ, etc.) followed by further flow cytometry purification [27]. The disadvantages associated with using predetermined marker(s) to identify CSCs include that the
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marker proteins frequently change during cell development in vivo and cell preparation in vitro and that in many cases the functions of these markers in both normal SCs and stem-like cancer cells are unclear. 2.2.2 Side Population (SP) Analysis Mouse HSCs are found to preferentially express multidrug resistance (MDR) family proteins such as MDR1 and other membrane transporters such as ABCG2 (also called breast cancer resistance protein (BCRP)) [28]. This property allows the HSCs, in an experimental setting, to pump out the Hoechst 33342 dye. Therefore, on dual-wavelength flow cytometry, the CSC-enriched cell population is identified as a side or tail Hoechstdim population at the lower left quadrant of the histogram. By contrast, the major population of cells is displayed as Hoechsthi cells called as non-SP or main population [29, 30]. Recent work reveals that multiple adult tissue SCs can also be enriched by the SP protocol and that the SP from several cancer types are also enriched in stemlike cancer cells [31, 32]. The major advantage of this technique when used to identify putative CSCs is its simplicity and independence of predetermined markers. The potential problems associated with the technique are that chronic accumulation of Hoechst dye in non-SP cells may be cytotoxic (thus invalidating suitable controls) and that SP cells isolated from some normal tissues or tumors seem to be enriched in progenitor cells rather than SCs. 2.2.3 Sphere Formation Assays Many normal SCs such as neural, hematopoietic, and mammary SCs, when maintained under special culture conditions, can form three-dimensional spheres, which are like mini-organs that can differentiate into multiple cell types. Putative CSCs identified in brain and prostate tumors as well as in melanoma also have the ability to form anchorage-independent spheres [24, 33, 34]. The advantage of using sphere-forming assays to enrich for CSC is its initial independence of specific markers. The disadvantages include the empirical nature of finding culture conditions suitable for sphere formation and the necessity of finding ways later to identify and purify the real CSCs from the spheres. 2.2.4 Label-Retaining Properties Mammary SCs and normal keratinocyte SCs in interfollicular epidermis and hair follicle bulges are quiescent and can be identified with a pulse label with the thymidine analog bromodeoxyuridine (BrdU) or H2B-GFP followed by a longterm ‘‘chase’’ (i.e., removal of the label). Fast proliferating progenitor cells dilute out the BrdU label after several cell divisions whereas the slow-dividing SCs retain BrdU and are thus identified as ‘‘label-retaining cells (LRCs)’’ by either immunohistochemistry or flow cytometry analysis [13, 35]. Interestingly, the LRCs in human breast tumors coexpress mammary epithelial SC markers
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and seem to have certain SC properties [36]. Human PCa [37] and nasopharyngeal carcinomas [38] also possess slow-cycling LRCs. The main challenge of this approach is that the LRCs have to be purified out live to show they indeed represent slow-cycling CSCs, which generally is difficult to do without a suitable genetic-tracking system. Furthermore, recent studies in HSCs suggest that not all LRCs may represent SCs [39]. On the other hand, the authors in this latter study [39] failed to show whether all the cells had been labeled with BrdU when the chase period began, as a very recent study shows that the H2B-GFP strategy labels a higher percentage of cells than using BrdU and that the label-retaining ovarian surface epithelial cells indeed possess certain SC properties [40].
2.2.5 Clonal Assay The study in primary human keratinocytes has revealed cellular heterogeneity with respect to their ability to form a clone related to different cell sizes – small cells could form clones but large cells would undergo terminal differentiation [41]. Further work has shown that three different types of clones possess different proliferative capacities [42]. The holoclone contains tightly packed small cells with greatest replicative capacity. In contrast, the paraclone is a loosely packed clone of large cells with a short replicative life span. The third type of clone, the meroclone, contains a mixture of cells of different proliferative potential and is a transitional stage between the holoclone and the paraclone [42]. Similar to primary keratinocytes, several head and neck cancer-derived cells can also form three different types of clone in culture [43]. More importantly, the holoclones but not paraclones highly express some stem cell-associated molecules, suggesting that holoclones might contain self-renewing stem-like cells [24, 43]. Our work, in several long-cultured cell lines of prostate, breast, and other cancer cells, confirmed that only a small fraction of cells can form holoclone in culture [24]. Our subsequent study in PC3 cells has revealed that holoclones are enriched for potential CSCs that can initiate serially transplantable tumors [44].
2.3 CSC Niche Normal SCs are found to harbor in a specialized microenvironment called the stem cell ‘‘niche’’, which contains capillaries, vascular endothelial cells, pericytes, and the fibrous proteins of the extracellular matrix. Other stromal cells, immune cells, and nerves may also be present in the niche [45]. Stimuli from the niche as well as signals from SCs themselves and from outside of niche, together, establish a regulatory network to balance between SC self-renewal and differentiation [46]. Under physiological conditions, the niche provides a tight control over proliferation, typically maintaining cells in G0 and/or balancing
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proliferation and apoptosis such that the SC population remains at a constant size [13, 47, 48]. Recent investigations in HSCs have uncovered two distinct niches, an osteoblast niche and a vascular niche [49, 50]. Osteoblasts lining the endosteal surface of the bone may function as the ‘‘quiescent or dormant niche’’, whereas the endothelial cells lining the bone marrow (BM) and spleen sinusoids may comprise the ‘‘activated or vascular niche’’ that regulates HSC expansion and differentiation. There has been some controversy over whether or not CSCs actually require specific niches. A recent study indicates that specialized microenvironments of BM (i.e., periendosteal region) seem to be required for the homing and engraftment of not only normal HSCs but also leukemic SCs [51]. CD133+ brain CSCs also seem to ‘‘live’’ in a vascular niche that secretes factors that promote their long-term growth and self-renewal [52]. Interestingly, CSCs themselves not only are dependent on factors produced by the vasculature to maintain self-renewal and long-term growth but also generate vascular epidermal growth factor (VEGF) and other factors to induce angiogenesis to promote vascular formation [52]. Another recent study also reveals that the CD133+ glioma SCs secrete more VEGF to enhance vascular formation compared to bulk cells [53]. In this case, CSCs and angiogenesis can positively feedback each other to promote tumor development and maintenance. Intriguingly, normal SC niches may also be involved in the metastatic process, as tumor cells disseminated from the primary tumor often migrate and seed microanatomical areas characteristically occupied by somatic SCs [46]. Disseminated CSCs might acquire responsiveness to certain secreted niche signals and thus initiate metastasis from these supportive microenvironments distant from the site of primary tumor [54]. The tumor microenvironment in a secondary organ may be considered as a ‘‘metastatic niche.’’ It is believed that the metastatic niche either passively supports tumor development or facilitates tumor formation. It has also been hypothesized that CSCs may remain dormant until activated by ‘‘appropriate’’ signaling from the microenvironment [55, 56]. Taken together, the potential role of the CSC niche in controlling the CSC fate provides some support for the theory that targeting the unique aberrant microenvironment of CSCs may represent a critical aspect of effective cancer therapy.
3 Chemotherapy and Drug Resistance Traditional chemotherapeutic strategies, on the assumption that the majority of the cells within tumor are actively proliferating, have used a variety of drugs and hormonal agents that interfere with the basic cellular machinery (e.g., DNA synthesis and replication, cell cycle, and cytoskeleton) [57]. However, because cancer cells share many properties with their normal counterparts, the serious, sometimes life-threatening, side effects that arise from toxicities to sensitive normal cells limit the efficacy of cytotoxic chemotherapy [58]. Improved understanding of the molecular alterations present in the cancer cells has enabled the
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development of a more cancer-specific ‘‘targeted therapy’’ for some types of cancer [57]. Such therapeutics aim at selectively targeting proliferating cancer cells and leaving normal cells untouched, thereby reducing the common side effects. Nevertheless, all therapeutics, either targeted or non-targeted, are designed to ablate proliferating cancer cells [58], which may lead to the possibility of tumor recurrence or drug resistance due to the presence of quiescent cells or slow-dividing cells in the tumor. Since chemotherapy became one of the main therapeutic strategies in cancer treatment, resistance has often followed. A number of studies indicate that there are three major mechanisms of drug resistance in cells: first, decreased uptake of water-soluble drugs such as folate antagonists, nucleoside analogs, and cisplatin, which require transporters to enter cells; second, various changes in cells that affect the capacity of cytotoxic drugs to kill cells, including alterations in cell cycle, increased repair of DNA damage, reduced apoptosis, and altered metabolism of drugs; and third, increased energy-dependent efflux of hydrophobic drugs that can easily enter the cells by diffusion through the plasma membrane [59]. Of these mechanisms, the one that is most commonly encountered is the increased efflux of a broad class of hydrophobic cytotoxic drugs, mediated by energy-dependent transporters, known as ATP-binding cassette (ABC) transporters, which includes seven subfamilies labeled A–G [60–62]. Interestingly, resistance occurs not only to traditional chemotherapeutic drugs but also to targeted therapeutics such as gefitinib (epidermal growth factor receptor or EGFR inhibitor), imatinib (an inhibitor of translocated BCR-ABL kinase in chronic myelogenous leukemia), and tamoxifen (targeting the estrogen receptor or ER in breast cancer) [63–65]. How do cancer cells acquire resistance to chemotherapy? Are there cells in the tumor, e.g., quiescent or slow-dividing cells that are naturally resistant to chemotherapy? Or the proliferating cells targeted by anti-cancer drugs acquire genetic changes to be resistant? The CSC hypothesis may bring us new insight on these important questions related to drug resistance.
4 CSCs in Chemoresistance and Radioresistance Based on the CSC hypothesis, CSCs may be naturally resistant to therapeutic agents. First, most chemodrugs target proliferating cells while CSCs are thought to be generally quiescent; therefore, CSCs will be mostly spared of drugs. Solid experimental data in support of this contention are yet to be provided. Second, the putative CSC niche may protect CSCs from damage during therapeutic treatment. Third, the ABC transporters that pump out drugs are preferentially expressed in CSCs, as described above for the SP cells. Many studies have shown that the SP phenotype is mediated by MDR proteins and that the SP tumor cells confer high drug efflux capacity [66–68]. A small population of stem-like cells in small-cell lung carcinoma has also showed coexpression of CD44 and multidrug resistance protein, MDR1,
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suggesting that CSCs may be drug-resistant [69]. Furthermore, one recent study indicates that melanoma cells positive for ABCB5 are much more tumorigenic than the corresponding ABCB5 cells and that ABCB5 actually mediates drug resistance in melanoma, suggesting that the ABCB5+ cells may represent melanoma stem cells [70]. Fourth, chemodrugs often exert their effects by eliciting cancer cell apoptosis, and overexpression of anti-apoptotic molecules such as Bcl-2 has been observed in some CSC populations [71]. Indeed, prospectively purified CSCs from human colon cancer specimen are more resistant to apoptosis than bulk cells [72]. Fifth, SCs are also thought to be more resistant to DNA-damaging agents than differentiated cells because of their ability to undergo asynchronous DNA synthesis and the enhanced capacity for DNA repair. During asynchronous DNA synthesis, the parental ‘‘immortal’’ DNA strand always segregates with the new SC rather than with the differentiating progeny, thus helping to protect the SC population from DNA damage [73–75]. Similarly, CSCs seem to be more resistant to DNAdamaging agents [76, 77]. Several lines of evidence indicate that CSCs may be intrinsically drugresistant. For example, putative CSCs are found to contribute to cisplatin resistance in a Brca1/p53 mutant mouse mammary tumor model [78]. In glioblastoma, the CD133+ CSCs demonstrate significant resistance to chemotherapeutic agents including teozolomide, carboplatin, paclitaxel, and etoposide in vitro [79]. In breast cancer xenografts, after several cycles of administering anti-cancer drugs to mice, the percentage of CD44+CD24 CSC population increases significantly. Furthermore, these cells exhibit much higher clonogenic and tumorigenic capacity than the parental cells [38]. Importantly, breast CSCs are enriched in breast cancer patients after administering chemotherapy [38]. Besides being ‘‘naturally’’ resistant to therapeutics, some CSCs may also acquire further resistance through accumulating mutations following exposure, producing a population of multidrug-resistant tumor cells that can be found in many cancer patients with recurrent diseases [80].
5 Novel Cancer Therapies by Targeting CSCs and Their Microenvironment One of the major questions raised by CSC hypothesis is whether or not current therapies are in fact targeting the right cells. As discussed above, it has been speculated that CSCs have the ability to avoid or resist current cancer therapies, although this has yet to be definitively proven in the clinical setting. In addition, CSCs may play an important role in tumor metastasis and dormancy. So, the idea that cancers could be effectively treated by selectively targeting CSCs and their microenvironmental niche, which contributes to self-renewal of these cells, has attracted tremendous clinical interest [81].
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5.1 CSC-Targeted Therapy As molecular mediators of therapeutic resistance in CSCs gradually become established, developing clinically useful inhibitors to target these pathways will be correspondingly prioritized.
5.1.1 Anti-ABC Transporters A connection between CSCs and drug resistance is thought to exist due to the expression of ABC transporters. Inhibition of ABC transporter activity should hinder CSC drug resistance and make CSCs more sensitive to chemotherapeutics. A number of inhibitors targeting ABCB1 including verapamil, cyclosporine, PSC833, and VX710 have been identified [80, 82]. However, negative results have been observed in clinical trials of these drugs possibly because other transporters, particularly ABCG2, also need to be efficiently inhibited to achieve a significant effect in the clinic. At present, tariquidar, effective against ABCB1 and ABCG2, is being tested, but some phase III clinical trials were terminated early owing to an increased incidence of adverse effects [83]. Selective downregulation of MDR genes in cancer cells is an emerging approach in therapeutics. Technologies that enable the targeted regulation of genes, including antisense oligonucleotides, hammerhead ribozymes, and shortinterfering RNA, have produced mixed results. However, sufficient downregulation of MDR genes such as MDR1 has proved difficult to attain and the safe delivery of constructs to cancer cells in vivo remains a challenges [59]. Interestingly, recent studies have revealed the possibility of using the agents targeting the EGFR, Hedgehog, Wnt/b-catenin, and/or Notch cascades to inhibit the ABC multidrug efflux transporters and/or eliminate the CSCs [84–90]. For instance, in the cyclopamine treatment for CD44+ PC3 metastatic prostate cancer cells, a decrease of the expression levels of MDR1/ABCB1 and BCRP/ ABCG2 was observed, suggesting that Hedgehog signaling activity may contribute to MDR expression in these cancer cells [89]. Since cancers rely on several mechanisms to escape drugs, ABC transporter inhibitors will have to be combined with other strategies to achieve efficient elimination of CSCs in vivo.
5.1.2 Inhibiting DNA Repair Capacity CSCs may efficiently repair their DNA to escape from DNA damage, raising the possibility that inhibiting DNA repair capacity of CSCs could be an effective strategy to sensitize CSC to current therapy. Recent studies in human glioblastomas have shown that checkpoint kinase 1 (Chk1) and checkpoint kinase 2 (Chk2) play a key role in CD133+ glioma stem cell survival to radiation by efficiently repairing damaged DNA; consequently, inhibition of Chk1/Chk2 kinases renders these CSCs less resistant to radiation [77]. These
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observations suggest that selective suppression of DNA repair mechanisms may help overcome radioresistance of CSCs. 5.1.3 Promoting Apoptosis Although tumor cell death is controlled by several mechanisms, one of the dominant mechanisms involves mitochondria [91]. It is thus rational to target the mitochondria for therapeutic purposes due to its critical role in the regulation of apoptosis. One potential target is the mitochondrial permeability transition pore (MPTP), the most significant component of which appears to be the peripheral benzodiazepine receptor known as the translocator protein [92]. Various ligands for the translocator protein have shown both anti-proliferative and pro-apoptotic activity or may act as chemo-sensitizers via modulation of MPTP [93–95]. In addition to mitochondria, other prosurvival signaling pathways such as nuclear factor kappa B (NFkB) and BCL-2 family proteins may also become targets for cancer therapy as CSCs generally have higher activities or expressions of these pathways. 5.1.4 Sensitizing Dormant CSCs to Anti-proliferative Agents Many types of cancer can persist as ‘‘minimal residual disease,’’ and putative metastasis SCs can remain dormant for years, but are re-activated by as-yet unknown mechanisms, often leading to rapid disease progression [96]. One possible strategy to sensitize dormant CSCs to anti-proliferative agents would be to promote their cell-cycle entry using cytokines known to activate normal SCs. For example, imatinib treatment of chronic myelogenous leukemia (CML) CSCs intermittently activated with granulocyte-colony stimulating factor (GCSF) in vitro resulted in an enhanced efficacy of CML–CSC elimination compared with treatment using imatinib alone [97]. However, to utilize this strategy in the clinic, new markers have to be developed to determine whether a patient has dormant disseminated disease. At present, most of the markers inform about the recurrence of the disease (e.g., uPAR, ErbB2) rather than the state of tumor cell dormancy [98]. To achieve this goal it will be important to enhance collaborations between basic research and clinical labs to test the markers of dormancy identified in experimental models in samples from bone marrow, lymph nodes, and circulating tumor cells (CTCs) [98]. These will be important for staging of cancer and for developing novel therapeutic targets. 5.1.5 Inducing CSCs into Dormancy Therapies aimed at inducing dormancy may help overcome conventional drug resistance that is based on the ability of drugs to induce cell killing. Thus, reprogramming cancer cells into a growth arrest would allow converting the disease that would be otherwise untreatable, into a chronic asymptomatic condition. Combination of mitogenic signaling inhibitors (e.g., MEK, MET,
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EGFR, IGFR inhibitors) with activators of stress pathways (e.g., p38, JNK) may induce reprogramming of tumor cells into dormancy [99].
5.1.6 Promoting Differentiation of CSCs The tumorigenic potential of CSCs generally correlates with their capacity to undergo long-term self-renewal [46]. Self-renewal activity is inversely correlated to differentiation, raising the possibility that enforced differentiation of CSCs could be an effective strategy to decrease or eliminate CSCs activity. This type of therapeutic strategy has been used to treat hematological malignancies such as leukemia, where the cancer-initiating cells and the cellular differentiation hierarchy are well characterized [100, 101]. The first differentiation agent successful in the clinic was all-trans-retinoic acid (ATRA) in the treatment of acute promyelocytic leukemia (APL). This strategy has recently been reported in human glioblastoma, where it was shown that bone morphogenetic proteins (BMPs) promote the differentiation of not only normal neural progenitors, but also of CD133+ glioma SCs toward a more mature astrocyte fate [102]. These findings should lead to renewed interest in devising therapies that promote the differentiation of cancer (stem) cells. Although differentiation therapy does not kill cancer cells, it does have the potential to restrain their self-renewal capacity and perhaps increase the efficacy of conventional therapies, which are often most effective on differentiated cells. Furthermore, differentiation agents often have less toxicity than conventional chemotherapies [103, 104].
5.2 CSC Niche-Targeted Therapy In light of the central role of niche in maintaining quiescence and self-renewal of CSCs, strategies for reduction of the SC population must take into account the importance of targeting niche. HSCs need hypoxic niche to protect them from oxidative stress because reactive oxygen species induce p38-MAPK-mediated proliferation leading to HSC exhaustion [105]. Some CSCs may also be concealed in hypoxic microenvironments, which are common in solid tumors. Given that hypoxia seems to be important in maintaining CSCs, this provides an opportunity for tumor-selective therapy. Hypoxia-inducible factor 1 (HIF-1) is stabilized under hypoxic conditions and promotes survival of cancer cells in hypoxic niches, so several strategies to inactivate HIF-1 are currently under investigation at the preclinical level [106, 107]. Other niches comprising reticular and endothelial cells known to secrete factors that promote self-renewal and survival have been found in close association with the vasculature [108]. Since the niche is dependent on the presence of an efficient blood supply, one therapeutic strategy would be to target the niche endothelium. Clinical use of anti-angiogenic agents has accelerated in
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recent years, with more than 40 agents currently in clinical trials for various types of cancers [109]. Anti-VEGF monoclonal antibody, bevacizumab, has shown promise as part of a combination therapy regimen in several advanced cancers, including colon cancer [110] and glioblastoma [111]. Moreover, several agents that were originally developed to block EGFR (erlotinib, cetuximab, and vandetanib) have recently been shown to have an inhibitory effect on angiogenesis by blocking VEGFR or by inhibiting pro-angiogenic protein secretion [109]. Furthermore, several lines of evidence have revealed the potential benefit of targeting the tumor-specific vascular precursor cells including the circulating VEGFR-2+ endothelial progenitor cells (EPCs) and VEGFR-1+ pleural mesothelial cells (PMC), which may be recruited to the activated stromal compartment in the primary and secondary neoplasms, for preventing and/or counteracting the neovascularization process associated with tumor development [112–115]. More particularly, it has been shown that the use of BMderived EPCs engineered to express an anti-angiogenic gene product, a soluble truncated form of VEGFR-2, may impair tumor growth in vivo [115]. There are several theories regarding the clinical mechanisms of antiangiogenic drug benefit. One possibility is that anti-angiogenics simply destroy the vascular structure of the tumor, promoting profound tumor hypoxia and nutrient deprivation. Alternatively, anti-angiogenics may transiently normalize the tumor vasculature increasing oxygen and drug delivery [116]. In addition, it seems that some cancers may express VEGF receptors as well, raising the possibility that anti-VEGF therapies actually have direct anti-tumor effects. Cell adhesion molecule CD44 is expressed on both normal and leukemic SCs and mediates adhesive interactions with the endosteal BM niche by binding to various ligands [117]. Therefore, it is possible to separate the leukemic SCs from its microenvironment by anti-CD44 therapy. In support, monoclonal antibody ligation of the CD44 effectively eliminated AML SCs derived from some patients [118]. Also, treatment of prostate and breast cancer cell lines with an siRNA against CD44 can decrease cancer cell adhesion to BM endothelial cells [119]. It is well known that signaling molecules such as Hedgehog, Wnt, BMP, fibroblast growth factor (FGF), and Notch-1 play important roles in niche control of cell fate determination [120], making it possible to develop novel cancer therapeutics by targeting these signaling pathways. Some studies have shown that the Hedgehog and Notch-1 signaling activities can be attenuated via Hedgehog inhibitor cyclopamine and Notch-1 inhibitor GSI, respectively [86, 121].
5.3 Combination Therapy Strategy A few considerations make combination therapy necessary in clinic. First, due to inherent genomic instability, differentiated cancer cells may acquire SC phenotypes, and also possibly acquire resistance to therapeutics. Thus, eradication of
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the tumor will likely be achieved only by successful targeting of all cancer cells. Furthermore, most of CSC- or niche-targeted therapies aim at sensitizing CSCs to current therapy or inhibiting CSC activity including anti-apoptosis and chemoresistance to cytotoxic drugs, not killing CSCs directly. Finally, the fact the tumor cells survive anti-tumor therapeutics via variegated mechanisms suggests that single targeting therapy would not effectively eradicate the disease. In one word, in order to eliminate CSCs and cure cancer, it seems reasonable that combining traditional therapy with one or several agents that sensitize CSCs or target CSC niche would be a good approach to rationally advance the treatment of tumors. For example, depletion of blood vessels by anti-VEGF therapy could cause a dramatic reduction in the number of medulloblastoma SCs, but this regimen had little effect on the proliferation and survival of nonCSC tumor progenitors. The combination of anti-angiogenic drugs and conventional therapies could be highly cooperative [122].
6 Conclusions and Future Directions Cancer recurrence and metastasis are major challenges of cancer therapy. Tumor dormancy may be one of the major reasons of cancer relapse and metastasis, and due to resistance, a subset of cancer cells that survives therapy may become the origin of cancer recurrence. The revived CSC hypothesis provides us new insight on understanding cancer cell resistance to therapeutics, especially to chemotherapy. CSCs are defined as a minor population of cancer cells that possesses the capacity to self-renew and to cause the heterogeneous lineages of cancer cells that comprise the tumor. Several experimental strategies such as utilizing specific markers, SP analysis, clonal and clonogenic assays, and LRC tracking can be utilized to identify or enrich for putative CSCs. Some evidence suggests that CSCs might play key role in cancer metastasis and dormancy. Like normal SCs, CSCs might be endowed with higher intrinsic capability to escape chemotherapy through their relative quiescence, ABC transporter expression, dysregulation in apoptotic signaling and increased prosurvival mechanisms, and their capacity for DNA repair. CSC niche is the specific microenvironment in which CSCs grow. It provides critical factors for the long-term survival and self-renewal of CSCs. Because CSCs are supposed to contribute to tumor development and metastasis as well as relapse after traditional therapies, it is of a great clinical potential that novel therapeutic strategies selectively target CSCs and their microenvironmental niche. Many clinical trials of drugs targeting bulk cancer cells or CSCs and their niches, although exciting and effective in preclinical trials, have not yielded positive results in patients. There are two possible reasons to explain this discrepancy. First, the preclinical models may be inappropriate. Numerous agents have shown very promising activity in animal models but have minimal clinical activity. One of the main reasons for this failure is the use of
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inappropriate mouse models for preclinical assays which are not CSC-based mouse models. So, the development of strategies to target CSCs and their niches will require new approaches for the preclinical evaluation of their efficacy [58]. In addition, drugs targeting CSCs also possibly affect normal SCs, with which CSCs share many properties. Because we use much of the knowledge gained about normal SCs to design new strategies against CSCs, most of these ideas center around attacking the stem-like properties of CSCs, and normal SCs may also be targeted in the process [54]. For example, blocking ABC transporters, such as ABCG2 and ABCB1, may make CSCs more sensitive to chemotherapy, but may also cause the body’s normal SCs to become sensitive to drugs and die prematurely. So, more work is needed with regard to delineating the similarities and differences between normal SCs and CSCs in order to identify possible therapeutic strategies that would effectively and specifically eradicate CSCs while leaving normal SCs unharmed. Acknowledgment We thank all current and past Tang lab members for their support and helpful discussions. We apologize to those colleagues whose original work could not be cited in this chapter due to space constraint. This work was supported in part by grants from NIH (R01-AG023374, R01-ES015888, and R21-ES015893-01A1), American Cancer Society (RSG MGO-105961), Department of Defense (W81XWH-07-1-0616 & W81XWH-08-1-0472), Prostate Cancer Foundation, and Elsa Pardee Foundation (D.G.T) and by two Center Grants (CCSG-5 P30 CA166672 and ES07784). JQ was supported by a post-doctoral fellowship from DOD and HY was supported by a fellowship grant from the Chinese Ministry of Education.
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