Concise reviews: cancer stem cells: from concept to cure

CANCER STEM CELLS Concise Reviews: Cancer Stem Cells: From Concept to Cure K. B. MATCHETT, T. R. LAPPIN Key Words. Cancer stem cells • Hematologic malignancies • Self-renewal • Differentiation

Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast, United Kingdom Correspondence: Professor T. R. Lappin, PhD, FRCPath, Centre for Cancer Research and Cell Biology, Queen’s University Belfast, Belfast BT9 7BL, U.K. e-mail: [email protected] Received July 10, 2014; accepted for publication July 14, 2014; first published online in STEM CELLS EXPRESS July 30, 2014; available online without subscription through the open access option. C AlphaMed Press V

1066-5099/2014/$30.00/0 http://dx.doi.org/ 10.1002/stem.1798

ABSTRACT In 1953, noting a remarkable consistency between the agents causing mutations and those associated with cancer, Carl Nordling, a Finnish-born architect, proposed that cancer results from an accumulation of genetic mutations. It is now generally accepted that inherited mutations and environmental carcinogens can lead to the development of premalignant clones. After further mutations, one cell reaches a critical state which confers a survival or growth advantage over normal cells. Such cells have the ability to initiate a malignant tumour. They share many of the features of normal stem cells, including the capacity for self-renewal and differentiation, and are widely termed cancer stem cells (CSCs). Although CSCs have been well characterized in hematological malignancies, their existence in some other tissues has been questioned. Here, we review recent work in which stem cells and stem cell-like cells have been used to investigate the pathogenesis of cancer and potential anticancer treatment strategies, in the context of both hematological and somatic tissue disease. STEM CELLS 2014;32:2563–2570

INTRODUCTION Advances in stem cell biology and the need to find cures for cancer patients have created a burgeoning global research effort in which stem cells are used extensively to investigate the pathogenesis of cancer and potential anticancer treatment strategies. The theory that cancer results from an accumulation of genetic mutations was first proposed by Nordling in 1953 [1] and further refined by Ashley, Knudson, and Nowell (reviewed in [2]). It postulates that inherited mutations and environmental carcinogens can lead to the development of premalignant clones, and that after further mutations one cell reaches a critical state which confers a survival or growth advantage over normal cells. This abnormal cell is then capable of initiating the formation of a malignant tumor. Due to their longevity stem cells have been widely held to be the subset of cells most likely to accumulate the number of mutations required to generate a cancer cell. The concept of cancer stem cells (CSCs) is predicated on the existence of normal tissue stem cells, which can either undergo division to produce new stem cells, or differentiate into more specialized cells. Although the term “cancer stem cell” is widely used in the literature, consensus on a universal definition for CSCs is lacking. Recently, however, Nguyen et al. have defined CSCs as “the cells within a

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malignant clonal population that can propagate the cancer under the assumption that these cells must be eradicated to achieve cures” [3]. This definition not only attests to the clonal nature of cancers arising from multiple sequential genetic and epigenetic events, and the phenotypic heterogeneity of tumors, but also recognizes that not all the cells within the malignant population have stem cell-like properties. Recent work demonstrating stem cell plasticity in mammary tumors indicates that the concept of CSC hierarchical organization may not hold for all tumors and underlines the need for independent investigations of tumors originating in different tissues [3]. Nonetheless, many cancers appear to be organized hierarchically and to harbor a small number of CSCs capable of renewing tumor growth, a property lacking in their progeny. This review will focus on recent CSC research in the context of hematological and somatic tissue malignancies, and progress in the characterization of signaling pathways, transcription factors, and other molecules involved in tumorigenesis.

HEMATOLOGICAL MALIGNANCIES CSCs have been well characterized in hematological malignancies. The stem cell origin of chronic myeloid leukemia (CML) was confirmed some 20 years ago when several groups

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identified and isolated CML cells capable of expansion in vitro, based on the characteristics that delineate normal hematopoietic stem cells (HSCs) [2]. Hematopoietic stem cell transplantation (HSCT) is a potentially curative option for patients with acute myeloid leukemia (AML). Better understanding of the biology of leukemic stem cells, and the practice of risk stratification using cytogenetics and molecular markers, has enhanced the selection of patients most likely to benefit from allogeneic transplantation. In their concise review, Hamilton and Copelan discuss the theoretical aspects of transplantation, analyze clinical data, and provide recommendations for the use of HSCT in AML [4]. AML and the myelodysplastic syndromes arise from transformed immature hematopoietic cells following the accumulation of multiple genetic and epigenetic perturbations in hematopoietic stem cells and committed progenitor cells. Pandolfi et al. review recent advances in the understanding of this series of transforming events that initially gives rise to preleukemia stem cells which precede the formation of fully transformed leukemia stem cells [5]. Although focal adhesion kinase (FAK) activity is implicated in many cancer phenotypes, little is known about its role in AML. FAK splice variant expression is deregulated in primitive leukemic cells in AML patients with poor prognosis. Targeting FAK was found to efficiently eliminate leukemic cells in vitro suggesting that FAK may be a useful therapeutic target for improved treatment of this subset of AML patients with poor prognosis [6]. LIM domain only 2 is one of the most frequently overexpressed oncogenes in human acute T-cell lymphoblastic leukemia (T-ALL). Analysis of CD2-Lmo2 transgenic thymic progenitor cells revealed that they were blocked in differentiation, quiescent, and immortalized in vitro on OP9-DL1 stromal cells. These cellular events are consistent with a transcriptional signature in Lmo2 transgenic T-cell progenitor cells that is also present in HSCs and in early T-cell precursor ALL [7]. The TEL-AML1 fusion gene often arises before birth creating a persistent preleukemic clone capable of converting to precursor B-cell leukemia after the accumulation of secondary genetic mutations. TEL-AML1-mediated self-renewal is associated with a transcriptional program shared with embryonic stem cells (ESCs), a finding that may provide a basis for targeting the TEL-AML1 transcription program [8]. The Friend erythroleukemia virus has been a long-standing model system for investigating the multistage process of leukemia progression and the mechanisms that regulate erythroid development. Building on earlier work indicating that Friend virus activates the BMP4-dependent stress erythropoiesis pathway, Paulson’s group showed that the two stages of Friend virus-induced disease are caused by infection of specific stress progenitor populations in the spleen, thereby establishing a new model for Friend virus-induced leukemia and demonstrating its utility as a model system for leukemia stem cell selfrenewal [9]. In two recent articles Bonnet’s group presented a new tool to identify chemoresistance and self-renewal mechanisms in primary human AML-initiating cells [10] and Ichim reported on kinase-independent mechanisms of resistance of leukemia stem cells to tyrosine kinase inhibitors [11]. Recent developments in diagnostic and therapeutic approaches have improved the progression-free survival rates in Hodgkin’s Lymphoma. Among these are functional imaging, refinement of clinical prognostic factors, and the development C AlphaMed Press 2014 V

of novel markers which facilitate improved patient selection and timing for autologous stem cell transplantation [12]. Premature ovarian failure occurs in a high proportion of patients who receive conventional chemotherapy or myeloablative therapy with HSCT. The authors of a phase II study involving 60 patients who underwent either myeloablative or nonmyeloablative regimens reported that the gonadotropinreleasing hormone analog, leuprolide, did not preserve ovarian function, highlighting the need to investigate other approaches to protect ovarian function [13].

BREAST CANCER Two interesting papers report on radiation effects in breast CSCs (BCSCs). Lagadec and colleagues found that ionizing radiation reprogrammed differentiated breast cancer cells into induced BCSCs that showed enlarged mammosphere formation, increased tumorigenicity, and expressed the same stemness-related genes as BCSCs from nonirradiated samples. The authors concluded that radiation contributes to the increased BCSC numbers seen after classic anti-cancer treatment [14]. Vieira et al. found that P-cadherin confers resistance to x-ray-induced cell death and mediates stem cell properties in basal-like breast cancer [15]. Key aspects of the role of BCSCs on the pathogenesis of breast cancer are examined in articles devoted to breast cancer initiation, progression, and therapy [16], the regulation of BCSCs by global signaling networks [17], mevalonate metabolism [18], and a novel role for tumor-associated macrophages in the regulation of a recently identified paracrine EGFR/ Stat3/Sox-2 signaling pathway [19]. Using a novel mouse mammary CSC model, epithelial tumor-initiating cells that express both basal and luminal mammary cell lineages were found to retain the potential to generate heterogeneous tumors. This may eventually enable the identification of therapeutic agents to target particular breast cancer subtypes [20]. State-of-the-art intravital imaging techniques have demonstrated remarkable stem cell plasticity in mammary tumors [21] and enabled the assessment of the remodeling of endogenous mammary epithelium by breast cancer cells [22]. Woodward’s group treated the aggressive breast cell lines SUM159 and MDA-231, as well as primary breast cancer cells derived from patients, with the histone deacetylase (HDAC) inhibitors valproic acid or suberoylanilide hydroxamic acid and found increases in aldehyde dehydrogenase (ALDH) activity, mammosphere-forming efficiency, and tumor-initiating capacity of non-stem-like cells, mediated through the WNT/bcatenin signaling pathway. Thus, at least in vitro, HDAC inhibitors can promote the expansion of breast CSCs through dedifferentiation, which may have important clinical implications [23]. Phosphosulindac (OXT-328), a novel derivative of the nonsteroidal anti-inflammatory drug sulindac, has been reported to selectively target BCSCs both in vitro and in human breast cancer xenografts [24]. In preclinical models of triple-negative breast cancer, the c-secretase inhibitor PF03084014 showed synergistic effects with docetaxel through multiple mechanisms, indicating that it may have the potential to enhance taxane therapy [25]. Elimination of tumorinitiating cells by inhibiting the p90 ribosomal S6 kinase through inactivation of the Y-box binding protein-1 in tripleSTEM CELLS

Matchett, Lappin negative breast cancers [26] and inhibition of EZH2, which is required for both breast and pancreatic CSC maintenance [27], may also offer rational approaches to therapy for some types of breast cancer.

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Several lines of investigation indicate that glioma stem cells (GSCs) contribute to therapeutic resistance in high grade glioma, and that eradication of GSCs is essential to achieve complete therapeutic response. To gain insights into the pathogenesis of high grade gliomas and glioblastoma multiforme, Nakano’s group investigated the role of MELK in GSCs [28, 29]. Rai has been characterized as a new regulator of neural progenitor migration and glioblastoma invasion [30] while CD133 has been found to be essential for the maintenance of GSCs [30]. Cellular plasticity is reported to confer migratory and invasive advantages on a population of GSCs that invade peritumoral tissue [31]. Numb regulates GSC fate and growth by deregulating EGFR and SCF ubiquitin ligase activity [32] and IGF1 receptor signaling regulates adaptive radioprotection in GSCs [33]. REST regulates the oncogenic properties of glioma cells [34] and the tumorigenic potential of miR-18A* in GSCs requires NOTCH-1 signaling [35]. Lee et al. report that inhibition of polo-like kinase 1 (PLK1) kills glioblastoma multiforme brain tumor cells partly through loss of SOX2 and delays tumor progression in vivo [36]. Other groups have demonstrated that Connexin 43 reverses the malignant characteristics of GSCs by regulating E-cadherin [37] and that cancer cell heterogeneity in glioblastoma can be counteracted by targeting the cytosolic innate immune receptors RIG-I and MDA5 [38]. Interestingly, GSCs can be targeted by metformin activation of FOXO3 via AMPK [39]. Yuan and colleagues found that GSCs exhibited low mitochondrial respiration and high glycolytic activity. The GSCs were sensitive to the glycolytic inhibitor 3-BrOP but resistant to conventional therapy such as carmustine and temozolomide (TMZ). Combination of 3-BrOP with carmustine, but not with TMZ, was strikingly synergistic and killed GSCs through depletion of cellular ATP and inhibition of the repair of carmustine-induced DNA damage, leading to impaired sphere-forming ability in vitro and enhanced survival of mice inoculated with GSCs [40]. In clinical trials, oncolytic viral therapies have shown promise for the treatment of glioblastoma. Sgubin et al. found that oncolytic Herpes simplex virus counteracts the hypoxiainduced modulation of glioblastoma stem-like cells, suggesting that further development of this approach is warranted [41]. Werbowetski-Oglivie et al. have shown that neural precursors derived from transformed human ESCs (N-t-hESCs) exhibit features of human brain tumors, and so can provide a model system to investigate the initiation and progression of primitive human neural cancers that are difficult to investigate using somatic sources [42]. CD133 is highly expressed in the CSCs of glioblastoma multiforme and appears to be associated with resistance of the CSCs to treatment with radiation and chemotherapy. In studies designed to target CD133 by cytotoxic T cells (CTLs) the Yu group cultured short peptide fragments of CD133 with dendritic cells that function as antigen-presenting cells in the immune system. Two potential human leukocyte antigen (HLA)-A*0201-restricted

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CD133 epitopes, CD133–405 and CD133–753, showed potential as anti-CSC antigens in GBM. Autologous monocyte-derived dendritic cells pulsed with either of the epitopes stimulated CTLs to efficiently recognize the CD133 epitopes presented in T2 HLAA*0201 cells, and to specifically lyse CD1331 HLA-A*02011 GBM CSCs. Immunization of mice with the murine homologs of the CD133 epitopes demonstrated immunogenicity in the absence of autoimmune damage. The study supports the use of CD133specific epitope vaccines to target CSCs in glioblastoma, and argues for a clinical trial to assess the safety of this approach [43].

COLORECTAL CANCER In a concise review of colorectal cancer (CRC) stem cells Vaiopoulos et al. discuss a recently introduced CSC model in CRC, biomarkers used for CSC identification, and various novel therapeutic approaches [44]. Screening of new anticancer drugs is generally performed on conventional tumor cell lines but this approach is controversial because the cell lines may not recapitulate CSC populations in primary tumors. Muraro et al. tested a panel of 10 established CRC cell lines and found that CD1331, CD1661CD441, and CD241CD441 phenotypes failed to reliably identify cell populations with stem cell functional features [45]. In an effort to identify CSC markers with greater specificity than CD44 and CD133, Medema’s group developed monoclonal antibodies against the Wnt target Lgr5 and showed that Lgr5 is a selective marker for human CRC stem cells [46]. Separately, Lgr5-positive CRC stem cells have been reported to undergo a reversible transition to drug-resistant Lgr-negative cells and to have the potential for tumor reconstitution [47]. Embryonic NANOG (NANOG1) is an important regulator of pluripotency and its expression is positively regulated by cJUN and b-catenin/TCF4. These two factors together have been postulated to drive a subpopulation of CRC tumor cells that adopt a stem-like phenotype via the NANOG1-promoter [48]. Colon cancer cells are dependent on PLK1 for proliferation [49], consistent with its essential role in glioblastoma multiforme brain tumor cells in tumor progression in mice [36]. In the quest to reduce metastatic progression from primary CRC, a vaccine targeting CSCs was found to efficiently reduce both tumor volume and occurrence of liver metastases in a rat colon carcinoma model [50].

MELANOMA The existence of melanoma stem cells has been widely debated. Two recent papers provide strong evidence that a population of cells with high ALDH activity are, in fact, melanoma stem cells. Luo and colleagues investigated human melanoma cells that fulfill the criteria for CSCs, namely selfrenewal and differentiation, by serial xenotransplantation into NOD/SCID mice. These cells possess high ALDH activity and are more tumorigenic than ALDH-negative cells in murine models. The gene signatures of the melanoma CSCs include retinoic acid-driven genes and those implicated in stem cell function [50]. Santini and colleagues used nonadherent spheres and ALDH enzymatic activity to enrich for CSCs in a collection of human melanomas. They found that C AlphaMed Press 2014 V

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melanospheres express high levels of Hedgehog pathway components and embryonic pluripotent stem cell factors. Pharmacological inhibition of Hedgehog signaling by the SMOOTHENED (SMO) antagonist cyclopamine, and the GLI antagonist GANT61, and targeting either SMO or GLI1 by shRNA resulted in a significant decrease in melanoma stem cell self-renewal in vitro accompanied by a reduction in the number of ALDHhigh melanoma stem cells [51]. The Rhodamine 123 (Rh123) exclusion assay has been used to identify stem-like cells in metastatic human melanomas and melanoma cell lines. A small subset of Rh123low cells was enriched for stem cell-like activities, including the ability to self-renew and produce non-stem Rh123high progeny from melanospheres, recapitulating the phenotypic profile of the parental tumor. Inhibition of the PI3K/AKT pathway caused specific reversion of a subset of Rh123high cells to the Rh123low phenotype that were less quiescent and displayed a significant increase in melanosphere formation-forming ability [52]. Anaka and colleagues analyzed the growth properties, transcriptional profile, and genotype of melanoma cells grown de novo in stem cell media (SCM) and found a neural lineage gene expression profile that was not representative of matched patient tissue specimens. They concluded that SCM culture of melanoma results in a neural bias that could potentially confound target identification [53].

PROSTATE CANCER Stem cell-like prostate cancer cells are capable not only of tumor initiation and maintenance in prostate cancer (PC) but also of tumor reinitiation in castration-resistant PC (CRPC). Germann and colleagues reported the identification of the human counterpart of murine castration-resistant cells (CARNs), which express the putative prostate tumor suppressor Nkx-3-1, luminal markers such as cytokeratin 18 and the androgen receptor, and possess stem cell-like properties. These human PC SC-/CARN-like cells may represent the cellof-origin of tumor reinitiation in CRPC and have potentially important clinical implications [54]. Taylor et al. used prostatic basal cells enriched based initially on a2b1 integrin (hi) expression and subsequently for stem cells by CD133. Unexpectedly the tumorigenic potential did not reside in the CD133(1) population but was consistently found in the CD133(2) population, confirming that benign human basal cells include cells-of-origin of prostate cancer and reinforcing their importance as therapeutic targets [55]. The TRAMP (transgenic adenocarcinoma of the mouse prostate) model is well characterized. TRAMP mice consistently develop a progressive lesion known as prostatic intraepithelial neoplasia that develops into adenocarcinoma between 24 and 30 weeks of age. Galli’s group established long-term self-renewing PCSC lines from the different stages of TRAMP progression using the tumor (or floating) sphere assay. These stage-specific prostate cell lines had CSC properties with well-defined gene signatures corresponding to distinct stages of human tumor progression. They should provide a valuable preclinical model for elucidating the pathogenetic mechanisms in prostate adenocarcinoma and for the identification of possible therapeutic targets [56]. C AlphaMed Press 2014 V

SARCOMA Soft tissue sarcomas may arise from mesenchymal stem cells (MSCs). The expression pattern of MSC markers in sarcoma cell lines, primary tumor samples, and fibroblasts has been examined by flow cytometry using a recently assembled panel of antibodies. Expression of W5C5, W8B2 (also known as tissue nonspecific alkaline phosphatase, TNAP), CD344, and CD271 marked subpopulations with increased proliferation potential, but the bona fide progenitor cell markers CD117 and CD133 were not expressed. Overall the data implied a hierarchical cytoarchitecture of the most common adult type sarcomas and establish W5C5, TNAP, CD344, and CD271 as potential sarcoma progenitor cell markers [57]. Sarcomas characterized by the presence of tumor-specific fusion oncogenes as a result of chromosomal translocations have been modeled in mice. Rodriguez et al. describe the first model of sarcoma based on the expression of a sarcomaassociated fusion protein FUS-CHOP in human MSCs, which should enable insights into the mechanisms of cellular transformation in sarcoma and testing for potential therapeutic targets [58].

OVARIAN CANCER Factors affecting intratumoral heterogeneity, invasion, and migration of ovarian cancer cells have been reported. By examining clonal subpopulations from a single ovarian clear cell carcinoma in a hESC-derived cellular microenvironment in mice, Abelson and colleagues demonstrated marked intratumoral phenotypic heterogeneity. Moreover, the derived cells displayed microenvironment-dependent plasticity with the capacity to switch from CD441/ALDH1 selfrenewing cells that sustain tumor growth to differentiated CD442/ALDH2 cells, leading the authors to propose that a paradigm shift is required in the approach to anticancer therapy [59].

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Angiogenic Factors The angiogenic properties of tumor-initiating cells (TICs) have been examined in a range of tumor types including breast (MCF7), glioblastoma (U-87MG), colon (HT29), non-small-cell lung (A549), and pancreatic (PAN1) cancers. Long-term cultures grown as monolayers (TIC-low) or as nonadherent tumor spheres (TIC-high) were generated. TICs from U-87MG and HT29 but not from MCF7, A549, and PANC1 possess increased angiogenic activity. Differential angiogenic activity and sensitivity to antiangiogenic therapy were found in the same tumor type, suggesting that the efficacy of antiangiogenic drugs, such as VEGF-A neutralizing antibody, is dependent on the angiogenic properties of the TICs [60]. Examination of the biological effects of MSCs on tumor cells showed that they inhibited the growth of human glioma cell lines and patient-derived primary glioma cells in vitro [61]. Coadministration of MSCs and glioma cells caused a STEM CELLS

Matchett, Lappin significant reduction in tumor volume and vascular density. Endothelial progenitor cell recruitment and the capacity to form endothelial tubes were also significantly impaired in conditioned media derived from MSC/glioma coculture, suggesting that MSCs suppress tumor angiogenesis through the release of antiangiogenic factors. Decreased expression of platelet derived growth factor (PDGF)-BB in MSC/glioma coculture implies that the antitumor effect of MSCs may be mediated through the PDGF/PDGFR axis which has an established role in glioma angiogenesis. MSCs have considerable potential as tumor-targeting vehicles due to their innate tumortropic homing properties. Keung et al. [62] review recent progress in novel cell-based cancer therapies using genetically engineered MSCs, in particular their application to targeting angiogenesis and tumor stroma in gastrointestinal (GI) malignancies, such as hepatocellular carcinoma and pancreatic adenocarcinoma. In a comprehensive proteomic analysis of human glioblastoma stem cells (GSCs) and neural stem cells, hepatomaderived growth factor (HDGF) has been identified as a novel angiogenic secreted factor [63]. GSC-conditioned medium induced migration of human cerebral endothelial cells that could be blocked with anti-HDGF antibodies in vitro, and neoangiogenesis, which could be abrogated by HDGF-targeted siRNAs in vivo.

Chemokine Receptors Long and colleagues compared a CD1331 specific population of ovarian cancer cells (CSLCs) to CD1332 non-CSLCs. In the former cell type C-C type chemokine receptor (CCR) 1, CCR3 and CCR5 were upregulated and blocking of CCR5, CCR1, or CCR3 effectively inhibited their invasive capacity, suggesting that activation of CCR1, CCR3, or CCR5 may contribute to the metastatic properties of ovarian CSLCs [64]. A subpopulation of the renal carcinoma cell (RCC) line RCC-53 expresses the CXC type chemokine receptor 4 (CXCR4). When grown as spheres, most of cells were CXCR4positive, expressed stem cell-associated transcription factor genes at elevated levels, and were more resistant toward the tyrosine kinase inhibitors sunitinib, sorafenib, and pazopanib. Interestingly, higher CXCR4 mRNA levels in primary RCC from patients with localized, but not disseminated, disease predicted shorter survival. More effective treatments of metastatic RCC may develop from combining standard treatment protocols with blockade of the CXCR4 signaling pathway [65].

Polycomb Group Proteins The polycomb group (PcG) proteins are composed of two multimeric protein complexes that function as epigenetic gene silencers in many aspects of development and in cancer. Polycomb recessive complex 1 includes the BMI1 polycomb ring finger, which promotes neural stem self-renewal. The role of BMI1 in recurrence and resistance in cancer has been reviewed [66]. The interaction of BMI1 with FoxG1 in the regulation of self-renewal and tumorigenicity of medulloblastoma stem cells has been investigated [67]. Both FoxG1 and BMI1 were significantly enriched in non-Shh/Wnt medulloblastomas of groups 3 and 4 that are characterized by metastatic progression, poor patient outcome and the lack of a molecular pathway phenotype.

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SOX Transcription Factors Two members of the Sox family of transcription factors have recently been implicated in CSCs. SOX2 is a self-renewal factor in lung stem cells and is highly expressed in a subpopulation of lung cancer cells in which proliferation, survival, and chemoresistance are dependent on SOX2 signaling. SOX2 elicits oncogenic EGFR and BCL1 signaling, and the expression of SOX2, EGFR, and BCL1 levels was significantly correlated in lung tumors. SOX2 is associated with poor prognosis in lung cancer and has potential applications as a prognostic marker and as a therapeutic target in the disease [68]. In the CD44high/ESAhigh subpopulation of cells sorted from head and neck cancers inhibition of GSK3b reduces the expression of Sox2, Oct4, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin [69]. Investigation of the epigenetic regulation of genes in the tumor-initiating population of pancreatic cancer cells revealed that SOX9, which is demethylated in CSCs, plays a critical role in the invasion process. The NF-ŒB signaling pathway is activated in pancreatic CSCs and inhibition of the pathway disrupted the stem cell-like properties of the cells. This study establishes a link between the canonical NF-ŒB signaling pathway and the invasiveness of pancreatic CSCs and could lead to the identification of novel molecules that function at the epigenetic level and which may form future therapeutic targets [70].

Hedgehog and Notch Signaling Pathways Aberrant activation of the Hedgehog and Notch pathways via mutations or overexpression has been implicated in many tumor types through pathogenetic mechanisms ranging from tumor initiation to angiogenesis and CSC maintenance. The polypeptide ligand Hedgehog (Hh) activates the signal transduction pathway that bears it name. The Hh pathway is essential for embryonic development, but is silenced in adults. Evidence is accumulating that it plays an important role in tissue repair and carcinogenesis. Promising results were obtained in early phase clinical trials of Hh inhibitors as monotherapy for patients with basal cell carcinoma and medulloblastoma, leading to the realization that combination therapy regimes and the development of correlative biomarkers should be prioritized in future research [71]. A close link has been found between the Notch 1 pathway and tumor vascularization of GBM cells [72]. Activation of Notch 1 signaling reduced the growth rate and migration of GBM cells, accompanied by a marked reduction in the expression of the neural stem cell transcription factors ASCL1, OLIG2, and SOX2 and by upregulation of HEY1/2, KLF9, and SNAI2. Following termination of Notch1 stimulation, expression of OLIG2 and growth were restored. In xenotransplantation experiments, Notch1-stimulated GBM cells resulted in poorly disseminated but highly vascularized tumors, in contrast to control GBM stem cells, which gave rise to infiltrative and poorly vascularized grafts. Notch1-stimulated GBM cells expressed pericyte cell markers and were closely associated with endothelial cells.

Transforming Growth Factor b1 Transforming growth factor b1 (TGF-b1) is a pleiotropic cytokine that mediates tumor growth, metastasis, and cytokine C AlphaMed Press 2014 V

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production. The combined effects of TGF-b1 and a hypoxic environment significantly induced self-renewal capacity in non-stem osteosarcoma cells, leading to increased chemoresistance, tumorigenicity, neovasculogenesis, and metastatic potential. In contrast, blocking the TGF-b1 signaling pathway inhibited the differentiation and clonogenicity of osteosarcoma cells and reduced CSC self-renewal capacity and hypoxia-mediated dedifferentiation. Thus, stem cells and non-stem cells exist in a dynamic equilibrium in this osteosarcoma cell population, and CSCs appear to develop de novo from differentiated cancer cells [73]. RUNX3 is a mediator of TGF-b1 and acts as an antagonist of Wnt. Loss of Runx3 in gastric epithelial cells results in spontaneous epithelial-mesenchymal transition (EMT) producing a stem cell-like subpopulation that remarkably expresses the gastric stem cell marker Lgr5. These results suggest that RUNX3 has a protective effect against abnormal growth factor signaling in gastric epithelial cells [74]. Treatment of colon and breast cancer cells with TGF-b1 triggered the EMT program and increased the expression of the large intergenic noncoding RNA, Hotair. Ablation of Hotair expression prevented TGF-b1mediated EMT and the colony-forming capacity of colon and breast tumor cells [75].

Pluripotency-Related Oncogenes Developmental pluripotency-associated 2 (DPPA2) regulates chromatin structure and functions in the maintenance of pluripotency and proliferation of embryonic stem cells. Another family member, DPPA4, is associated with active chromatin and is involved in the differentiation of ESCs into a primitive ectoderm lineage. DPPA4 is highly expressed in embryonal carcinoma, pluripotent germ cell tumors, and other malignancies. Interestingly, an expression screen for oncogenic foci-inducing genes in a human embryonic stem cell cDNA library identified DPPA4 and DPPA2 as oncogenes [76].

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CONCLUSIONS The renaissance of interest in CSC biology has led to a number of methodological advances for the study of CSCs. Mather has reviewed the selection of subpopulations of existing tumor lines derived from serum-containing media, the creation of lines from malignant or normal cells by genetic manipulation, and the direct selection of CSCs from tumors or sorted tumor cells using defined serum-free conditions [77]. Label-retaining cells (LRCs) are thought to arise from either slow-cycling or asymmetric cell division and to represent adult tissue stem cells. Using a novel method for the isolation of live LRCs, Xin and colleagues demonstrated label-retaining cancer cells (LRCCs) in several GI cancers, including fresh surgical specimens. Moreover, by realtime confocal microscopic cinematography they were able to track live LRCCs undergoing asymmetric nonrandom chromosomal cosegregation LRC division. The authors concluded that LRCC may represent a novel population of GI stem-like cancer cells [78]. The collection of recent papers reviewed here covers important aspects of the occurrence, pathogenesis, and treatment of a range of cancers. It is representative of a huge amount of literature covering CSCs spanning basic, translational, and clinical research and should provide a plethora of options for readers interested in current cutting-edge investigations in cancer and leukemia.

AUTHOR CONTRIBUTIONS K.B.M. and T.R.L. planned and wrote the article, contributed equally in the writing of the articles.

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The authors indicate no potential conflicts of interest.

7 Cleveland SM, Smith S, Tripathi R, et al. Lmo2 induces hematopoietic stem cell-like features in T-cell progenitor cells prior to leukemia. STEM CELLS 2013;31:882–894. 8 Tsuzuki S, Seto M. TEL (ETV6)-AML1 (RUNX1) initiates self-renewing fetal pro-B cells in association with a transcriptional program shared with embryonic stem cells in mice. STEM CELLS 2013;31:236–247. 9 Hegde S, Hankey P, Paulson RF. Selfrenewal of leukemia stem cells in Friend virus-induced erythroleukemia requires proviral insertional activation of Spi1 and hedgehog signaling but not mutation of p53. STEM CELLS 2012;30:121–130. 10 Griessinger E, Anjos-Afonso F, Pizzitola I, et al. A niche-like culture system allowing the maintenance of primary human acute myeloid leukemia-initiating cells: A new tool to decipher their chemoresistance and selfrenewal mechanisms. Stem Cells Transl Med 2014;3:520–529. 11 Ichim CV. Kinase-independent mechanisms of resistance of leukemia stem cells to tyrosine kinase inhibitors. Stem Cells Transl Med 2014;3:405–415.

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