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"\n\n\n\nThe survival of patients with cancer has improved significantly, primarily because of multidisciplinary care, improved chemotherapeutic agents in both the adjuvant and metastatic settings, the introduction of targeted biologic agents, and the incorporation of palliative care services into the management scheme. However, despite these advances, a significant proportion of patients continue to experience recurrence after adjuvant treatment, and survival associated with stage IV solid tumors still remains low. A primary or acquired resistance to chemotherapeutic and biologic agents is responsible for the failure of many of the agents used to treat patients with a malignancy. This can be explained by the presence of intratumoral heterogeneity and the molecular complexity of many cancers. Factors contributing to intratumoral heterogeneity include genetic mutations, interactions with the microenvironment-and the presence of cancer stem cells. Cancer stem cells have been identified in a number of solid tumors, including breast cancer, brain tumors, lung cancer, colon cancer, and melanoma. Cancer stem cells have the capacity to self-renew, to give rise to progeny that are different from them, and to utilize common signaling pathways. Cancer stem cells may be the source of all the tumor cells present in a malignant tumor, the reason for the resistance to the chemotherapeutic agent used to treat the malignant tumor, and the source of cells that give rise to distant metastases. This review will focus on properties of cancer stem cells; will compare and contrast the cancer stem cell model with the clonal evolution model of tumorigenesis; will discuss the role of cancer stem cells in the development of resistance to chemotherapy; and will review the therapeutic implications and challenges of targeting cancer stem cells, with an assessment of the potential such an approach holds for improving outcomes for patients with cancer.\n\nIntroduction\n\nIn the year 2014 an estimated 1,665,540 new cancer cases will occur in the United States, leading to approximately 585,720 deaths.[1] Over the last 2 decades, the combined cancer death rate (deaths per 100,000 population) has been on the decline, from a peak of 215.1 in 1991 to 171.8 in 2010. That translates to approximately a 20% decline in the death rate, with a reduction of 1,340,400 cancer deaths during this time. Factors contributing to the improved survival of patients with cancer include multidisciplinary care, improved chemotherapeutic agents in both the adjuvant and metastatic setting, the introduction of targeted biologic agents (eg, trastuzumab for breast cancer, imatinib for chronic myeloid leukemia [CML]), and the incorporation of palliative care services in the treatment management scheme.[2,3] However, despite these advances, a significant proportion of patients continue to experience recurrence after adjuvant treatment, and survival associated with stage IV solid tumors still remains low, with cure not a goal.\n\nOver the last few decades, the burning questions have been: why do tumors recur, and why is cure not considered an achievable goal in the metastatic setting? A primary or acquired resistance to chemotherapeutic and biologic agents is responsible for the failure of many of the agents used to treat patients with a malignancy. This can be explained by the presence of intratumoral heterogeneity and the molecular complexity of many cancers, such that while some of the tumor cells perish from exposure to chemotherapy, other cells survive exposure and contribute to disease recurrence and progression.[4] Factors contributing to intratumoral heterogeneity and ultimately acquired resistance to chemotherapeutic and biologic agents include genetic mutations, interactions with the microenvironment-and the presence of cancer stem cells.[4] Cancer stem cells are similar to normal stem cells in that they have the ability to self-renew and differentiate. They differ from normal stem cells in that the mechanisms that normally strictly regulate these processes are deregulated, such that there is continuous expansion and production of aberrantly differentiated progeny.[5]\n\nThe first compelling evidence for the presence of cancer stem cells came in 1997, when Bonnet and Dick demonstrated that only CD34+CD38− cells derived from patients with acute myeloid leukemia (AML) could initiate hematopoietic malignancies in nonobese diabetic/severe combined immunodeficient mice.[6] Since then, cancer stem cells have been identified in a number of solid tumors, including but not limited to breast cancer,[7] brain tumors,[8] lung cancer,[9] colon cancer,[10] and melanoma.[11] This review will focus on properties of cancer stem cells; comparison of the cancer stem-cell model with the clonal evolution model; discussion of the role of cancer stem cells in the development of resistance to chemotherapy; and a review of the potential therapeutic implications and challenges of targeting cancer stem cells, with an assessment of the potential to improve outcomes for patients with cancer.\n\nProperties of Stem Cells\n\nThree types of stem cells exist: embryonic stem cells (derived from the first division of a fertilized egg), which eventually give rise to all the cells in the adult organs; germinal stem cells (responsible for reproduction); and somatic stem cells (present in different somatic tissues). Normal stem cells have three distinct and unique properties: (1) they can renew themselves, which allows them to perpetuate themselves and maintain a pool of undifferentiated stem cells; (2) they can differentiate into multiple lineages and thus reconstitute a broad range of functional elements within any given tissue; and (3) they can maintain a balance between self-renewal and differentiation, thereby strictly regulating stem cell number. Multiple pathways are involved in the regulation of normal stem cells. The Notch and Sonic hedgehog pathways have been implicated in the regulation and differentiation of neuronal stem cells.[12] Other pathways typically associated with oncogenesis that have also been implicated in the regulation of stem-cell self-renewal include the octamer-binding transcription factor 4, bone morphogenic protein, Janus kinase, and Wnt signaling pathways.[13]\n\nWhat makes a cancer stem cell different?\n\nCancer stem cells comprise a small population of cells within a tumor. They are also known as “tumor-initiating cells” or “tumorigenic cells.” Cancer stem cells and normal stem cells have similar cell surface markers. Like normal stem cells, cancer stem cells have the capacity to self-renew, can give rise to different progeny, and utilize common signaling pathways.[14] They differ from normal stem cells in that they have tumorigenic activity that enables them to form tumors when transplanted into animals-something normal stem cells cannot do.[15] Cancer stem cells can be the source of: (1) all the tumor cells present in a malignant tumor; (2) resistance to the chemotherapeutic agent used to treat the malignant tumor, thus making them responsible for recurrence; and (3) cells that give rise to distant metastases.\n\nSeveral assays have been developed to isolate cancer stem cells. The most widely used is based on the presence of specific cell surface makers, and the most common methodology used to identify cell surface markers or intracellular molecules is the fluorescence-activated cell sorting method. Markers such as CD133, CD24, and CD44 are typically identified.[16] Table 1 provides examples of the cell surface phenotypes of cancer stem cells in different tumors. Leukemic stem cells have been shown to display the CD34+CD38− surface marker phenotype, in which the loss of CD38 distinguishes these cells from normal hematopoietic stem cells.[6] Breast cancer stem cells have been shown to display the ESA+CD44+CD24−/(low) surface marker phenotype.[7] However, this finding is not considered sufficient for cancer stem cell identification and is usually combined with results from functional assays such as sphere-forming assays (in which cancer stem cells form spheres or colonies in a serum-free or soft agar medium) or transplantation assays (which utilize the properties of self-renewal and tumor propagation inherent to cancer stem cells). In serial transplantation assays, tumor cells are transplanted into immunocompromised mice. Tumors that grow in these mice are then transplanted into another set of immunocompromised mice in order to exhibit the properties of self-renewal and capacity for tumor formation.[15,17]\n\nThe clonal evolution model vs the cancer stem cell model\n\nTwo separate and mutually exclusive models have been developed to explain the development of tumors. The clonal evolution model postulates that all cells within a tumor contribute in varying degrees to the maintenance of the tumor.[27] In this model, a number of genetic and epigenetic changes occur over time, with the result that the most aggressive cancer cells are ultimately responsible for driving tumor progression. Furthermore, through a series of genetic mutations, any cancer cell within the tumor can become invasive, lead to the development of metastases, and contribute to resistance to therapies and ultimately to recurrence of disease.\n\nThe cancer stem cell model proposes that cancer stem cells, which form a subset of the tumor cells, are ultimately responsible for tumor initiation, progression, and recurrence.[27] Through self-renewal and differentiation, cancer stem cells are responsible for the production of various tumor cells and contribute to tumor heterogeneity. Furthermore, according to this hypothesis, tumor metastases and resistance to therapies directly arise from cancer stem cells.\n\nBoth the clonal evolution and the cancer stem cell models have distinct therapeutic implications. In the clonal evolution model, a cure can only be achieved when treatment results in the death of the multiple tumor cell populations that are responsible for tumor progression. In the cancer stem cell model, a cure is only possible when treatment targets the cancer stem cells. For the remainder of this review we will focus on therapeutic strategies designed to target the cancer stem cell.\n\nRole of Cancer Stem Cells in Resistance to Chemotherapy\n\nCancer stem cells have been implicated in the development of resistance to chemotherapy in a number of malignances. Evidence for this emerged first in the setting of hematologic malignancies[6] and then in other solid tumor types.[7-11]\n\nHematologic malignancies\n\nPatients with AML are treated with courses of chemotherapy followed by consolidation therapy and autologous or allogeneic hematopoietic stem cell transplantation. Those patients who experience a complete response are still at high risk for recurrence caused by residual cells that survive chemotherapy exposure. Early studies have established that AML cells are hierarchically organized and contain CD34+ cancer stem cells that can sustain serial transplantation and have been implicated in chemotherapy resistance.[6] Van Rhenen et al[28] looked at the association between the presence of CD34+CD38− cancer stem cells and clinical outcome in 92 patients with AML. They found that a high percentage of these cancer stem cells at baseline correlated with a high frequency of minimal residual disease posttreatment and a poor prognosis. Furthermore, the minimal residual disease detected after complete response has been shown to be enriched with CD34+CD38− cancer stem cells, which are subsequently associated with relapse.[29] These cancer stem cells have also been shown to be resistant to therapy with cytarabine, with the resistant cells successfully engrafted into recipients.[30]\n\nCML, characterized by a translocation between chromosomes 9 and 22 that results in the oncogenic BCR-ABL tyrosine kinase, has been successfully treated with the tyrosine kinase inhibitor imatinib and second-generation inhibitors such as dasatinib and nilotinib. The presence of cancer stem cells in CML has been implicated in recurrence of disease following discontinuation of therapy, even among patients who achieve a long-term remission.[31-33]\n\nSolid tumors\n\nOne of the first solid tumors in which the presence of cancer stem cells was demonstrated is breast cancer. Al-Hajj et al[7] demonstrated that CD44+CD24−/(low)Lineage− cells isolated in eight of nine patients with breast cancer had the capacity to form tumors when serially transplanted into immunocompromised mice. They reported that as few as 100 cells with this phenotype were needed to form tumors, while thousands of cells with other phenotypes were unable to form tumors when transplanted. Ginestier et al[34] demonstrated that breast cancer cells with increased aldehyde dehydrogenase activity have stem cell properties and are associated with poor prognosis. Human epidermal growth factor receptor 2 (HER2) has also been shown to be an important regulator of breast cancer stem cells, with HER2 overexpression associated with an increase in the cancer stem cell population and blockade of HER2 associated with a decrease in the cancer stem cell population.[35]\n\nEvidence exists for the presence of cancer stem cells in colorectal cancer, with some phenotypes being CD133+[10] and CD44+/CD166+[23] enriched cancer stem cells. CD133+ enriched cancer stem cells have been shown to be more resistant to fluorouracil and oxaliplatin compared with CD133− cells.[36] Evidence indicates that pancreatic cancer stem cells are enriched with CD133+ and CD44+cMet+ cell surface phenotypes.[25,26] Patient-derived pancreatic xenografts treated with gemcitabine have demonstrated an increased frequency of cancer stem cells, which indicates resistance to the therapeutic agent.[37]\n\nBrain tumors\n\nSingh et al[19] have demonstrated the presence of CD133+ enriched cancer stem cells in glioblastoma (GBM) tumors. Furthermore, CD133+ cancer stem cells from GBM cell lines have been shown to be resistant to the chemotherapeutic agent temozolomide compared with CD133− cells.[38]\n\nTherapeutic Strategies Directed at Cancer Stem Cells\n\nTraditional and conventional modalities of treatment of various malignances include surgery, chemotherapy, treatment with biologic agents, and radiation therapy. Despite the variety of modalities available, resistance to treatment still occurs, resulting in recurrence and progression of disease, which according to the cancer stem cell model is attributed to the presence of a small subpopulation of cancer stem cells. Factors contributing to cancer stem cell resistance to therapeutic agents include the activation of signaling pathways that aid in self-renewal; the presence of multiple drug resistance membrane transporters, such as the ATP-binding cassette drug transporters; a capacity for DNA repair, and the microenvironment. All of these mechanisms are potential targets in efforts to eliminate cancer stem cells (Table 2).\n\nTargeting the cancer stem cell signaling pathways\n\nAberrant signaling pathways result in the formation of cancer stem cells, which are ultimately responsible for tumorigenesis. Aberrations of several important signaling pathways that are involved in regulating normal stem cell self-renewal, proliferation, and differentiation have been implicated. When aberrantly activated, the Hedgehog (Hh) signaling pathway, which is essential for the maintenance of stem cells, has been implicated in the tumorigenesis of various malignancies.[39] For example, genetic modulation of the Hh pathway has been shown to play a prominent role in CML pathogenesis by regulating the process of self-renewal of cancer stem cells.[40] Furthermore, using the Smoothened antagonist cyclopamine, which inhibits this pathway, has been shown to improve the efficacy of tyrosine kinase inhibitors, resulting in the depletion of cancer stem cells and subsequently in the improved survival of CML-bearing mice.[41]\n\nThe Notch signaling pathway, activated through ligand-receptor interactions of Notch receptors (Notch1 to Notch4) and Notch ligands (Delta1,3,4 and Jagged1,2), is important for stem cell proliferation, differentiation, and apoptosis. Its activation can either be oncogenic or oncosuppressive.[42] It is activated in a variety of malignancies, including breast cancers and malignant gliomas. Inhibition of this pathway with antibodies directed against Delta-like ligand 4 or Notch1 has been shown to reduce the population of breast cancer stem cells; this approach also results in improved efficacy of taxane therapy in patient-derived xenografts.[43-45] Gamma-secretase inhibitors have been shown to block the Notch signaling pathway, resulting in reduced expression of the putative markers of cancer stem cells, and subsequently in reduced tumor growth in vivo.[46]\n\nThe Wnt signaling pathways are also important targets. Of them, the Wnt/beta-catenin signaling pathway (canonical pathway) is the best characterized, with accumulating evidence indicating that it is involved in oncogenesis and tumor development.[47] Inhibitors of this pathway include small molecule inhibitors (nonsteroidal anti-inflammatory drugs and molecularly targeted agents such as the CREB-binding protein/beta-catenin antagonist ICG-001) and biologic inhibitors (antibodies, RNA interference, and recombinant proteins).[47] Recent evidence indicates that the Wnt/beta-catenin signaling pathway regulates cancer stem cell renewal in CML, and deletion of the beta-catenin results in a profound loss of residual cancer stem cells in the bone marrow of mice subjected to imatinib therapy.[48,49] Recent preclinical studies have shown that a combination of indomethacin (a cyclo-oxygenase inhibitor) and imatinib prolongs survival in CML transplantation models.[49]\n\nAnother important pathway that is currently being investigated as a potential target in a number of malignancies is the interleukin 8 (IL-8) signaling pathway, which regulates the activity of cancer stem cells by means of its cognate receptors CXC chemokine receptor (CXCR)1 and CXCR2. Therapeutics aimed at inhibiting CXCR1/2 signaling are thought to halt disease progression in tumors driven by IL-8.[50] Recent evidence indicates that IL-8 can induce a state of “stemness” by effecting epithelial-mesenchymal transition, which is needed for cells to acquire stem cell characteristics.[51] Activation of CXCR1/2 signaling in breast cancer cell lines has been shown to expand the pool of cancer stem cells.[52] Ginestier et al[53] showed that repertaxin, a noncompetitive inhibitor of CXCR1/2 signaling, reduced the proportion and activity of cancer stem cells in vitro-also that the in vivo treatment of mouse xenografts with reparixin resulted in increased efficacy of docetaxel, with subsequent decreased tumor growth.\n\nTargeting cancer stem cell surface markers\n\nA more specific therapeutic strategy is to target specific markers on the cancer stem cells. Table 1 gives examples of cell surface markers observed in cancer stem cells in different malignancies. Specific antibodies targeting these markers are currently under investigations.[54] CD 133+ cancer stem cells (present in a number of malignancies, including brain tumors and colorectal cancers) have been shown to be resistant to chemotherapy and radiotherapy, with high levels associated with poor prognosis.[36] Challenging GBM-CD133+ and GBM-CD133− cells using single-walled carbon nanotubes that were conjugated to a CD133 monoclonal antibody and then irradiated with near infrared laser light, Wang et al[55] demonstrated selective eradication of GBM-CD133+ cells. Using lentivirus-mediated short-hairpin RNA, Brescia et al[56] further demonstrated that silencing CD133 expression in human GBM neurospheres disrupts the self-renewal and tumorigenic properties of the neurosphere cells, indicating that CD133 could potentially be used as a therapeutic target in these tumors.\n\nIn human AML, antibodies against differentially expressed surface markers on cancer stem cells, such as CD44, IL-3R, and T-cell immunoglobulin and mucin domain–containing molecule 3 (TIM-3), have been shown to eradicate cancer stem cells and subsequently decrease leukemogenicity in mice.[57-59] CD47 stem cells are expressed at higher levels in acute lymphoblastic leukemia (ALL) than in normal cells. Therapeutic targeting of CD47 stem cells in ALL with a specific antibody has been shown to kill leukemia stem cells.[60]\n\nTargeting quiescence\n\nEvidence exists that quiescence, a property that keeps a cell in a nondividing state but allows the cells to retain the capacity to re-enter a cell cycle at a later time, is important in hematopoietic stem cells and may be a mechanism explaining resistance to chemotherapeutic agents.[61-64] One mechanism for overcoming chemotherapy and radiation therapy resistance is to induce quiescent cancer stem cells to enter proliferation in a cell cycle. Graham et al[63] demonstrated an enrichment of quiescent CD34+ cells when patient-derived CML cells were exposed to imatinib. Cell-cycle induction of these cells in vitro with granulocyte colony-stimulating factor (G-CSF) has been shown to improve the efficacy of imatinib.\n\nIshikawa et al[65] further demonstrated that exposure of AML stem cells to G-CSF enhanced the proliferative phenotype of these cells, which subsequently led to increased sensitivity to the chemotherapeutic agent cytarabine. Two phase III randomized clinical trials looking at the priming effect of G-CSF on outcomes in patients with AML reported favorable results, thereby providing clinical evidence that cell-cycle induction may improve efficacy of standard chemotherapy.[66,67]\n\nTargeting epigenetic mechanisms\n\nEpigenetic mechanisms, including DNA methylation, histone modification, and RNA-mediated targeting, are thought to be important regulators of cancer stem cells.[68] Histone deacetylases (HDACs) have been shown to regulate chemotherapy resistance in hematologic malignancies. In an in vivo study, Zhang et al[69] reported that using an HDAC inhibitor resulted in significant suppression of CML stem cells following imatinib exposure. Li et al[70] went on to demonstrate that in vivo selective sirtuin HDAC inhibition resulted in suppression of CML stem cell growth via a p53-dependent mechanism. MicroRNAs are also thought to be important epigenetic components of cancer stem cell regulation. For example, Liu et al[71] demonstrated that the microRNA miR-34a directly inhibits CD44, resulting in suppression of prostate cancer stem cells and metastasis.\n\nTargeting the tumor microenvironment\n\nCancer stem cells reside within a “stem cell niche” or microenvironment that consists of vascular, mesenchymal, and inflammatory cells; this microenvironment provides the signals necessary for regulating the properties of cancer stem cells.[72] CXCR4 is known to maintain bone marrow stem cells in the bone marrow microenvironment. Inhibition of CXCR4 has been shown to disrupt the interaction of CML cells with that microenvironment, which ultimately sensitizes them to nilotinib.[73,74] The perivascular microenvironment has been shown to regulate the tumor-initiating capacity of GBM cancer stem cells, and the microvascular endothelial cells within this environment have been shown to be responsible for the associated resistance of GBM cancer stem cells to chemotherapy, which results in treatment failure.[75,76] These examples reveal that targeting the cancer stem cell microenvironment as a therapeutic target is a potential strategy for overcoming resistance to therapy.\n\nTargeting cancer stem cell apoptosis\n\nDeregulation of apoptosis has been shown to be an important factor in reducing the efficacy of chemotherapy, with a growing body of evidence indicating that cancer stem cells are involved in promoting aberrations in apoptosis and thus ultimately in promoting resistance to therapy.[77] The mitochondrial B-cell lymphoma 2 (Bcl-2) family of proteins are known to regulate apoptosis. The anti-apoptotic protein Bcl-2 both protects hematopoietic stem cells from apoptosis and increases their potential for repopulation.[78] In an in vivo model of blast crisis CML, Goff et al [79] demonstrated a higher expression of Bcl-2 on bone marrow–derived cancer stem cells. When subjected to sabutoclax, a pan–Bcl-2 inhibitor, the blast crisis CML stem cells were sensitive to tyrosine kinase inhibitors (dasatinib). Bcl-2 overexpression has also been demonstrated in prostate cancer stem cells and breast cancer stem cells, where these cells play a role in promoting chemotherapy resistance.[80,81]\n\nTargeting metabolism\n\nDeregulation of the metabolic pathway is also known to play a key role in tumor development, with cancer stem cells exhibiting unique metabolic features.[82] Cancer stem cells in basal-like breast cancer have been shown to have distinct glucose and mevalonate metabolism, while cancer stem cells in lung cancer have increased expression of glycine decarboxylase.[83-85] Thus, the use of metabolic-specific drugs, such as metformin, that would target cancer stem cell metabolism may be a potential therapeutic strategy. Iliopoulos et al[86] reported that the addition of metformin improved the efficacy of paclitaxel and carboplatin in xenograft models derived from breast cancer cell lines and improved the efficacy of doxorubicin in xenografts generated from prostate and lung cancer cell lines.\n\nChallenges in Cancer Stem Cell–Directed Therapy\n\nThe elegant components of the cancer stem cell model and the extensive in vivo and in vitro data that support it indicate that cancer stem cells are a potential therapeutic target in a broad range of malignancies. However, for a number of important reasons, the applicability of this approach is limited. First, cell surface markers on cancer stem cells may be co-expressed on non–cancer stem cells; may vary in expression between patients with the same malignancy; and may exhibit plasticity through the course of the natural history of the disease, as well as through exposures to various therapeutic agents.[87] Second, cancer stem cells can also exhibit plasticity in their phenotypic features and functional properties, and we still have a long way to go before we have fully understood, defined, and elucidated cancer stem cell biology.[87] A full understanding of the biology of those cells would also be important to the development of the more standardized assays needed to help capture and detect the presence of cancer stem cells before we might appropriately target them. Third, the resistance of cancer stem cells to chemotherapeutic agents can vary between experimental models. In one model an agent may be toxic to the cancer stem cells, while in another model cancer stem cells may be resistant to the same agent. An example is the sensitivity and resistance to temozolomide exhibited in the in vivo models of GBM cancer stem cells.[88,89]\n\nFuture Directions\n\nSystemic therapies have demonstrated an inability to cure metastatic solid tumors, and typically patients die of progressive disease and resulting organ failure. Most current therapies have either a cytotoxic or cytostatic effect on cancer cells, but their ability to eliminate cancer stem cells is still unknown. The growing understanding of the biology of cancer stem cells is providing opportunities for detection, isolation, and therapeutic targeting of those cells. Our ability to incorporate cancer stem cell–targeting agents in the management of advanced epithelial malignancies should translate into prolonged benefit from systemic therapy, ultimately prolonging survival. 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