Discussing the Evolving Role of Cancer Stem Cells

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1 EXPERT PANEL LOCAL INSIGHTS Discussing the Evolving Role of Cancer Stem Cells Lee M. Ellis, MD Professor of Surgery, Department of Surgical Oncology William C. Liedtke Jr. Chair in Cancer Research The University of Texas MD Anderson Cancer Center Houston, Texas INTRODUCTION Besides understanding what gives rise to cancer, the major clinical challenges in managing these diseases include treatment failure or resistance, recurrence, and metastasis. Over the past several decades, increasing evidence has mounted for the existence of cancer stem cells (CSCs) and their involvement in these challenges. 1,2 Like normal stem cells, CSCs have the potential to self-renew and differentiate into progenitor cells, which further differentiate into tumor cells. 3 Normal stem cells are, however, under strict controls to limit these potentials. In general, they remain dormant except during normal tissue development or in response to injury. While CSCs share some of the main features of normal stem cells, the processes of proliferation and genetic repair are no longer subject to the usual control mechanisms. This creates a situation in which the CSC activation and differentiation potentials are altered. 3,4 Self-renewing, tumorigenic CSCs have been found in multiple tumor types and are usually resistant to standard therapies. These properties of CSCs make them suited to serve as the putative source of tumor origin as well as the source of tumor heterogeneity, recurrence, therapeutic resistance, and metastasis. 2,4 With the ultimate goal of developing more effective approaches to cancer management, researchers are endeavoring to gain a complete understanding of CSC biology. They are working to find ways to identify CSCs and to develop strategies that inhibit their stem-cell like properties and related signaling pathways, or that destroy them directly. The hope is that these approaches will help advance long-term cancer management. Local Insights has brought together clinicians and researchers to discuss the role of CSCs in cancer development and progression, and potential treatment approaches to interfere in these roles Boston Biomedical, Cambridge, MA All rights reserved. Printed in USA/December Jenny C. Chang, MD Emily Herrmann Chair in Cancer Research & Division Chief, Cancer Center Professor of Cancer, Institute for Academic Medicine Director Houston Methodist Cancer Center Houston, Texas Sendurai A. Mani, PhD Associate Professor, Department of Translational Molecular Pathology Co-Director, Center for Stem Cells & Developmental Biology The University of Texas MD Anderson Cancer Center Houston, Texas Supported by EDU-NPS-0063

2 LOCAL INSIGHTS: Discussing the Evolving Role of Cancer Stem Cells SECTION 1. What Are CSCs? Over 150 years ago, Virchow and others proposed that tumors, like other tissues, may arise from a subpopulation of cells with stem-cell like properties. 5 Only within the last 20 years, however, has proof of the existence of CSCs been demonstrated. 2 In 2006, the American Association for Cancer Research Workshop on Cancer Stem Cells established the definition of CSCs as cells within a tumor that possess the capacity to self-renew and to give rise to the heterogeneous lineages of cancer cells that comprise the tumor. CSCs are sometimes referred to in the literature as tumor-initiating cells or tumorigenic cells. 6 Today, CSCs have been identified in numerous hematologic and solid malignancies, including leukemia, lung cancer, colon cancer, prostate cancer, breast cancer, ovarian cancer, brain cancer, and melanoma. 2,7 CSCs interact with their tumor microenvironment to maintain stem-cell like properties, or stemness, that enable them to produce a variety of differentiated cells that compose the Figure 1. Normal stem cell and cancer stem cells (CSCs) development. Normal stem cells self-renew (curved green arrow) and differentiate into progenitor cells, which then differentiate into mature tissue. CSCs develop via mutation of normal stem cells or progenitor cells (dashed arrow), and can self-renew and differentiate into heterogeneous tumors. 8 majority of the tumor. 3 (See The Microenvironment on page 6.) A common misconception is that all CSCs arise from mutated normal stem cells, but some CSCs may arise from progenitor cells when a mutation endows these cells with the capacity for self-renewal, which is normally only the case with stem cells (Figure 1). 5,8 The Stochastic and Hierarchy Models of Tumor Heterogeneity Two main models exist to explain the development of heterogeneity within tumors: the clonal evolution or stochastic model and the CSC or hierarchy model. 9 Longheld theories about cancer, such as the stochastic model, posited that all cells within a tumor had essentially the same genetic changes and exhibited the same phenotype. Because it was thought that tumors were mostly homogeneous and that all tumor cells possessed tumor-initiating activity, cancer research was conducted on whole tumor populations, and treatments were developed keeping in mind the eradication of the tumor bulk. 10 In recent decades, observations of tumor heterogeneity and evidence for the existence of CSCs have presented a challenge to such models. The hierarchy model holds that minority subpopulations of biologically and functionally distinct cells have the capacity to initiate tumor growth and foster other tumor properties such as progression and aggressiveness. 4 Furthermore, these tumor-initiating cells can be identified and isolated from the bulk tumor. 10 While these models appear mutually exclusive, some have proposed that merging the models may actually provide a fuller explanation of tumor maintenance and progression. In fact, evidence indicates that aspects of the two models may describe tumor organization at different stages or under varying microenvironmental conditions. Such a unified model may account for the variation in the frequency, genotype, and phenotype of CSCs between patients and tumors. 2,11,12 CSC Properties Within both primary and metastasized tumors, cell subpopulations can differ on the basis of such factors as morphology, expression of surface antigens, 2

3 specific alterations of the genome, and patterns of gene expression Likewise, CSCs are heterogeneous with varying degrees of self-renewal capacity, development potential, and expression of cellular markers. Like normal stem cells, CSCs exist in a hierarchy. 5 Their capacity for self-renewal and differentiation places CSCs at the top of a cellular hierarchy from which all other cells within a tumor are derived. In this light, tumor origin and development may be likened to normal tissue development wherein stem cells differentiate into committed progenitor cells, and these further differentiate into the terminal cell types that constitute the bulk of the tissue. 16 The analogy to normal stem cells and normal tissue development weakens, however, in light of the fact that CSCs are cancerous. As noted, they are not subject to normal cell control mechanisms. Instead, CSCs undergo uncontrolled replication and differentiation, resulting in progenitor and terminal cell populations with altered molecular and cellular phenotypes. 2,4,13,17 A listing of the characteristics of normal stem cells and CSCs can be seen in Table 1. 3,4,13,18 Using glioma stem cells, research has shown that CSCs can divide symmetrically, producing new CSC progeny, or asymmetrically, producing non-stem cell and stem cell progeny. Intratumoral heterogeneity likely derives from asymmetrical division and differentiation of CSCs. 19 Over time, unrestrained differentiation and proliferation produces the heterogeneous populations of primary and metastatic tumor cells that contribute to tumor properties such as recurrence, resistance to therapy, and metastasis. 13 Table 1. Characteristics of Normal Stem Cells and Cancer Stem Cells 3,4,13,18 Normal Stem Cells CSCs Characteristic Self-renewal Differentiation Plasticity a Quiescence a Injury or experimental manipulation may induce plasticity in differentiated normal stem cell progeny Treatment Resistance CSCs tend to be resistant to chemotherapeutic agents and radiation. 20 The resistance of CSCs to standard therapies is due, at least in part, to their low rate of proliferation. 20 Following treatment, the remaining CSCs can repopulate the tumor, causing treatment failures and disease relapses. 13 Another feature of CSC biology and other tumor cells is that they possess inherent genetic and epigenetic instability. This instability is the foundation of tumorigenesis and tumor cell heterogeneity. It also implies that CSCs and non-cscs within a tumor can assume new phenotypes at different stages during the tumor life cycle. Thus, the cells are moving targets with the potential to develop treatment resistance over time. 6 CONSULTANT COMMENTARY Dr Chang: Simply, CSCs are a subpopulation of tumor cells that have repopulating potential, can self-renew, and are treatment resistant. Dr Mani: Cancer is made up of various cell types. Two important ones are differentiated cancer cells and CSCs. CSCs have been shown to be responsible for tumor relapse, acquisition of resistance to chemotherapy, and progression of disease to metastasis. Clearly, our group at MD Anderson and others have demonstrated that CSCs can be generated not only from the transformation of normal stem cells but also from the differentiated cancer cells by the activation of a latent embryonic program known as epithelial-mesenchymal transition. Dr Chang: In my mind, the hierarchical/csc and the clonal/stochastic theories are intertwined. They are not mutually exclusive. Even in clonal evolution, the ancestral clones appear to have more metastatic potential because they essentially have stem-cell like properties. Dr Ellis: We have the stochastic model, which is random, and the hierarchical model. The truth is somewhere between. Let s recognize that there s a whole spectrum of CSCs that differentiate themselves. The two extremes are probably stable and in between they re probably very plastic. 3

4 LOCAL INSIGHTS: Discussing the Evolving Role of Cancer Stem Cells SECTION 2. The Importance of CSCs Currently, conventional cancer treatments are intended to reduce bulk tumor burden by surgical resection 9,11,21 or by killing mature, rapidly proliferating tumor. 8,20 However, these treatments usually have little effect on CSCs, which are only a small portion of the total tumor mass. 13 As a result, patients often experience initial successful treatment responses that later give way to tumor recurrence, progressive disease, therapy resistance, and metastases. 13 Although chemotherapy and radiation therapy may eradicate much of the tumor bulk, theoretically, CSCs may remain, giving rise to treatment resistance. 13 In addition to resistance to many conventional treatment modalities, emerging evidence suggests that some current cancer therapies may actually enrich subpopulations of drug-resistant CSCs, increasing their numbers and their stem cell activity, and possibly, accelerating disease progression (Figure 2). 3,5,22 For example: Using human colon cancer cell lines, researchers found that treatment with two of the more commonly used chemotherapeutic agents caused the emergence of a drug-resistant subpopulation of cells expressing CSC markers CD24, CD133, CD44, and CXCR4. 1 In a mouse breast cancer model, ionizing radiation reduced the overall tumor volume but induced an increase in stem cell behavior as well as the expression of stemness genes and proteins. In addition, radiation treatment correlated with an approximate 50% increase in the nodular area of lung metastases compared to the controls. 23 Other research found that irradiation of breast cancer cells in vitro induced expression of Notch receptor family members that are known to be important in the self-renewal of breast and glioma CSCs. 24 Research has also indicated that the presence of CSCs within a tumor is associated with worse overall prognosis in many cases. 4,13 Functionally, CSCs have been found to be pivotal in such processes as angiogenesis, resistance to apoptosis, and cell migration and invasion. 13 As CSC biology is elucidated, increasing evidence suggests that effective tumor eradication will require the development of therapeutic regimens directed against CSCs, their underlying signaling pathways, and non-csc tumor cells. As much as possible, these therapies must still spare normal stem cells and healthy tissue. 2,6 Figure 2. Cancer stem cells (CSCs) and treatment resistance. Although current therapeutic regimens kill a significant amount of tumor cells, experimental evidence suggests that these regimens enrich therapy-resistant CSCs that are highly tumorigenic and may lead to tumor relapse. 3,5,22 4

5 The concept of a rare population of stem cells as the origin of cancer was first proposed over 150 years ago by Virchow and Cohnheim. 1 In the 1970s, researchers advanced the theory that tissue-specific stem cells might be the cells of origin for specific cancers, but technical advances were needed to provide experimental proof to support the CSC hypothesis. 1,2 Bonnet and Dick provided such evidence in 1997 by demonstrating the existence of leukemic-initiating cells that possessed differentiative, proliferative, and self-renewal capacities consistent with what was expected of CSCs. 3 In 2004, researchers in the same A Brief History of CSC Research group used cellular marking studies to show that the leukemic CSCs resembled normal stem cells in their degree of heterogeneity and their varying capacity for self-renewal. 4 Other researchers produced corroborative findings in breast CSCs. 5 Such findings have led to the hypothesis that CSCs are part of a developmental hierarchy that functions much like normal stem cells and that CSCs derive from malignant transformation of normal stem cells. 2 Subsequent research has continued to find CSCs in other tumor types, including those in the brain, prostate, and lung, as well as in multiple myeloma Huntly BJ, Gilliland DG. Leukaemia stem cells and the evolution of cancer-stem-cell research. Nat Rev Cancer. 2005;5(4): Wicha MS, Liu S, Dontu G. Cancer stem cells: an old idea a paradigm shift. Cancer Res. 2006;66(4): ; discussion Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997;3(7): Hope KJ, Jin L, Dick JE. Acute myeloid leukemia originates from a hierarchy of leukemic stem cell classes that differ in self-renewal capacity. Nat Immunol. 2004;5(7): Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Prospective identification of tumorigenic breast cancer cells. Proc Natl Acad Sci U S A. 2003;100(7): CONSULTANT COMMENTARY Dr Mani: A tumor is not static tissue. While you re treating a tumor today, it s evolving. CSCs appear to play a significant role in that process. In fact, CSCs have been shown to have a role in all types of cancer, both solid and hematologic. Dr Mani: Because of its chemical nature, chemotherapy can promote inflammation, which can induce plasticity. It s as though chemotherapy is angering the CSCs. Alternatively, chemotherapy could be killing differentiated cancer cells and enriching CSC populations. Dr Ellis: Theoretically, CSCs proliferate at a lower rate than more differentiated tumor cells. CSCs are hypothesized to mediate resistance to chemotherapies and, perhaps, targeted therapies. If, indeed, a subpopulation of CSCs exists, and they can be targeted or eliminated, the tumor should lose its source cell. This would allow better control of tumor growth or apoptosis. However, this is easier said than done. Identifying the multiple drivers of CSC-ness may provide a new opportunity for therapeutic targeting. 5

6 LOCAL INSIGHTS: Discussing the Evolving Role of Cancer Stem Cells SECTION 3. The Role of the Microenvironment and CSC Signaling Pathways CSCs grow and develop in a microenvironment that supports their survival, propagation, and differentiation into the cells that define the tumor mass. 14 In addition, CSC development is driven by a number of key signaling pathways and elements. 2 Dysregulation of the pathways and elements plays an important role in the ability of CSCs to self-renew and differentiate. Depending on the pathway involved, CSCs may initiate cancer formation or cause tumor recurrence. 2 The Microenvironment CSCs exist within a microenvironment of surrounding vasculature, stromal cells, immune cells, and secreted factors produced by these cells (Figure 3). 5,14 These create a niche wherein the CSCs can survive and thrive in order to propagate and differentiate into the cells that make up the tumor mass. In essence, the niche is a regulatory microenvironment that nurtures the stem-cell like characteristics of CSCs so that they can generate, or regenerate, the tumor bulk and maintain their self-renewing potential. The microenvironments that maintain normal stem cells, serve as a reserve for repopulating cell populations lost due to aging or damage. In the case of cancer development, these normal stem cell microenvironments may, however, become dysfunctional, hijacking their function to maintain CSCs. These CSCs may function as a tumor-cell reserve to foster recurrence following treatment. Evidence of this has been shown in brain tumors wherein CSCs were found with neighboring endothelial cells. Such an association resembles the usual niche for normal stem cells, which are the likely cells of origin for CSCs in these types of tumors. Furthermore, higher irradiation of a potential neural CSC niche improved survival among glioma patients. 9,12 Intracellular and intercellular signals operate within CSC microenvironments and support CSC activities. The internal signals include molecular pathways that regulate stemness, whereas extracellular signals consist of cells designed to anchor CSCs within the microenvironment, and cell receptors and secreted factors that are necessary for maintaining CSCs in their quiescent state. 25 Signaling Pathways Signaling pathways are key components in all cells. They stimulate a wide variety of cell processes from cell growth, proliferation, and differentiation to invasion and apoptosis. Well-known internal signals or pathways that function in normal stem cell niches include the Wnt, Notch, Hedgehog (Hh), and Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathways. 12,26 Several intracellular signaling pathways may be altered in the process of malignant transformation of stem cells (Figure 4). 2 For example: The evolutionarily conserved Wnt family of proteins are cysteine-rich, secreted glycoproteins that control tissue homeostasis, and regulates diverse processes during development. Wnt pathway dysregulation has been identified in several hereditary diseases and is associated with intestinal cancers. 27 Figure 3. Interaction of cancer stem cells (CSCs) with their microenvironment. CSCs exist and thrive within a microenvironment containing stromal cells, immune cells, neighboring vasculature, and factors secreted by these cells. These components create a niche where CSCs survive and propagate into a heterogeneous tumor mass. Courtesy of Boston Biomedical. 5,14 6

7 The Notch pathway has crucial roles in stem cell control and cell-fate determination. 28 Research has found that a signature of Notch pathway in CSCs identified patients with poorly differentiated lung adenocarcinoma, and was prognostic for poorer overall survival. By inhibiting the Notch pathway, CSCs were prevented from forming tumors when implanted into mice. 28 The Hh protein family members turn on the genes that regulate the cell cycle and determine cell fate. They are also known to be key regulators of carcinogenesis. Hh and downstream factors have been shown to have significant roles in pancreatic cancer, glioma, and basal cell carcinoma. Inhibition of the Hh pathway in pancreatic cancer depressed the self-renewal of CSCs and impaired their resistance to chemotherapy. 7,16,29 The Hippo pathway and its related mediators Yes/Yap regulate several tumor suppressor genes to control cellular processes such as survival, proliferation, differentiation, apoptosis, and stem or progenitor cell expansion. 1,30 Dysregulation of the Hippo pathway has been identified in multiple cancers including liver, lung, colorectal, ovarian, Figure 4. Key signaling pathways in cancer stem cell (CSC) development. Dysfunction of several developmental and stemness signaling pathways contribute to the generation of CSCs and their ability to initiate cancer formation and cause tumor recurrence. 2 and prostate. 30 Researchers also found that the expression levels of Yes/Yap genes were prognostic for survival in patients receiving certain types of chemotherapy. 1 NANOG is a transcription factor involved in the selfrenewal and maintenance of pluripotency in normal stem cells. Experimental inhibition of NANOG or related transcription factors have been shown to decrease stem-cell like activities in breast cancer, colorectal cancer, prostate cancer, and melanoma. 31,32 Several specific signaling pathways JAK/STAT, Wnt, Notch, Hh and Hippo have been shown to be involved in the induction and maintenance of CSC stemness. 2,31,35-40 The STAT family of transcriptional factors cooperates with NANOG to transcribe stemness genes that are required for modulating pluripotency. 33 The STATS are upstream signals activated by interleukin-6 (IL-6). 34 Activated STAT3 has been found in leukemia, squamous cell carcinoma of the head and neck, multiple myeloma, breast cancer, and prostate cancer. 26 Blockade of the STAT3 signaling pathway has been shown to inhibit the clonogenic and tumorigenic potential of CSCs in prostate cancer. 26 In addition, it has been shown that blockade of STAT3 activity inhibits both tumor growth and tumor-initiating potential in colon CSCs. 34 Cancer-associated cells in the microenvironment may secrete growth factors and cytokines to support CSCs. 11 Examples of these include cytokines such as stromal cell-derived factor-1, IL-6, and IL-8, all of which function to regulate CSC activity and are thought to play roles in treatment resistance. 11 For example, it has been shown that myofibroblasts within colorectal tumor-associated stroma secrete hepatocyte growth factor that maintains CSC function by activating the Wnt pathway. 9,40 Furthermore, research on glioblastomas has found that in some tumors there is a bidirectional relationship between the CSCs and the local microenvironment. These data suggest that elements of the microenvironment affect the cellular behavior of CSCs. Conversely, CSCs may be able to modify their microenvironment. 40 7

8 LOCAL INSIGHTS: Discussing the Evolving Role of Cancer Stem Cells Additionally, direct, cellular interactions between CSCs and other cells of the microenvironment function to preserve CSCs and affect their functions. In ductal breast carcinoma, elevated levels of the integrin molecule, focal adhesion kinase (FAK), have been linked with an increased invasive phenotype, and ablation of FAK function reduced the number of CSCs in a mouse breast cancer model. Many researchers have found further links between FAK signaling and breast CSC maintenance, tumor progression, and metastasis. 11 CONSULTANT COMMENTARY Dr Chang: Some cells have adaptive response to different stresses. Under different conditions, these cells will have different properties. We believe that the properties of CSCs can change depending upon the stresses, like chemotherapy and the microenvironment. Dr Mani: We re in the very early stage of understanding the whole biology of CSCs and developing therapeutics to inhibit CSCs and their signaling pathways. Dr Chang: From the clinical point of view, this is absolutely true. We are just starting to understand what triggers CSCs. We are developing therapies that target alterations in some of the signaling pathways, as well as cytokines involved in inflammation. It s an evolving field. Dr Ellis: A CSC is smart enough to know that it will need multiple pathways to maintain its stemness. Therefore, combination therapy will be crucial. SECTION 4. The Next Steps in CSC Research Identifying CSCs To distinguish CSCs from other cancer cells, researchers have developed profiles of unique cellular markers (Table 2). 35,36 These profiles allow detection of CSCs within a tumor and enable the separation of CSCs from non-stem cancer cells for research purposes. Several markers have proven useful for the isolation or enrichment for CSCs in multiple tumor types. These include CD133, CD44, epithelial cell adhesion molecule (EpCAM), and aldehyde dehydrogenase (ALDH) activity. It must be noted, however, that no set of markers are exclusive to CSCs, and also that CSC phenotypes vary over time and between individual patients tumors of the same subtype. These facts have caused researchers to speculate whether different Table 2. The CSC Markers Identified in Various Tumor Types 35,36 Tumor Type Phenotype of CSC Markers Breast cancer ESA +, CD44 +, CD24 /low, Lineage, ALDH1 high Brain cancer CD133 +, BCRP1 +, A2B5 +, SSEA1 + Colon cancer CD133 +, CD44 +, CD166 +, EpCAM +, CD24 +, CXCR4 +, CK20 +, CEA +, LGR5 +, ALDH1 high Head and neck cancer CD44 +, ALDH +, YAP1 +, BMI1 + Leukemia CD34 +, CD38, HLA-DR, CD71, CD90, CD117, CD123 + Liver cancer CD133 +, CD49f +, CD90 + Lung cancer CD133 +, ABCG2 high Multiple myeloma CD138- Pancreatic CD133 +, CD44 +, EpCAM +, CD24 +, ABCG2 high Prostate cancer CD44 +, α2β1 high, CD133 + Adapted by permission from MacMillan Publishers Ltd on behalf of Cancer Research UK: Chen K, et al. Acta Pharmacologica Sinica. 2013;34:

9 clinical outcomes reflect different CSC populations. 40 Following is a discussion of some common markers: CD133 is one of the most common cell surface marker used to identify CSCs. Originally identified as a marker for normal stem cells, CD133 is now also known to be present in CSCs from breast, prostate, pancreas, brain, and lung cancers. Studies have also shown that CD133 is useful not only for detection but also as a prognostic marker. 41 CD133 has also been shown to detect peritoneal free cancer cells in patients with surgically resected colorectal cancer. In addition, a combination of CD133 with other markers, CEA and CK20, was shown to be prognostic for survival in these patients. 42 However, other studies in Conflict Resolution Committee dispute the role of some of these markers in defining CSC populations. 43 In human leukemia, a combination of markers CD34, CD38, and IL3Ra were used to isolate leukemia CSCs. 40 Breast CSCs have been identified using a combination of markers, ESA +, CD44 +, CD24 /low44, and other groups have shown that elevated ALDH activity in both human and mouse breast cancer is a marker for CSCs. 23 Biomarker Tests Under Investigation Many researchers are investigating biomarkers to detect CSCs and/or assess their likely response to different treatments. Some of these findings are summarized below: Gastric CSCs expressing CD44 were resistant to 5-FU, etoposide, and radiation therapy. 45 Colon CSC lines compared with parental cell lines were resistant to 5-FU and expressed higher levels of CSC markers CD24, CD44, CD133, and CXCR4. 1 Prostate cancer cells exposed to a clinically relevant regimen of ionizing radiation, left a resistant subpopulation that showed increased expression of stemness markers CD133, SOX2, OCT4, and NANOG. 46 Figure 5. Combination of anti-cancer stem cell (CSC) and conventional treatments. Conventional cancer therapies appear insufficient to kill all tumor cells. A more rational approach may be to combine conventional therapies with those aimed at the CSCs and their signaling pathways. Such therapies may potentially decrease tumor recurrence and metastases. 13,20,38 Combination Therapy Approaches Multiple research findings indicate that conventional therapies that target the rapidly dividing cells in tumors have limited efficacy or even adverse effects on CSCs. 13 For example, in separate studies of pancreatic cancer tissues, expression of CD44 in CSCs was shown to correlate with resistance to chemotherapy and poor outcomes, whereas elimination of CD133 +, tumorigenic, chemotherapy-resistant CSC populations suppressed metastasis but did not affect the tumorigenic potential. Both these findings link CSCs to tumor aggressiveness. 13 Combining conventional therapy with CSCdirected therapy may lead to a reduction in overall tumor mass and a decrease in the risk of relapse and metastasis (Figure 5). 13,20,38 Such a multifaceted treatment approach might also prevent non-stem tumor cells from dedifferentiating back to a CSC phenotype, a phenomenon that has been demonstrated in cell lines. 11 9

10 LOCAL INSIGHTS: Discussing the Evolving Role of Cancer Stem Cells Two groups of researchers reported an increase in breast CSC numbers and activity induced by ionizing radiation therapy. In efforts to counteract this effect, the groups added additional therapeutic agents directed toward CSCs. One group added disulfiram plus copper, which is understood to affect the regulation of stemness genes via proteasome inhibition, 23 and the other added a γ-secretase inhibitor (to affect the Notch pathway). 24 Both combination regimens resulted in a reduction in the number of radiation-induced CSCs. 23,24 These and other data may provide justification for the development and incorporation of CSC-targeted therapy into treatment regimens to reduce the likelihood of treatment resistance, tumor aggressiveness, and tumor regrowth. They may reduce the tumor burden and ultimately improve outcomes of patients with CSC-possessing cancers. 13,38 CONSULTANT COMMENTARY Dr Mani: We do have several markers and assays available to study CSCs. What s missing is accurate ways to identify them. One challenge is that many CSC markers are shared by normal stem cells and are mostly expressed in particular tissues. For example, a marker used in breast cancer to identify a CSC may not be useful in a different tumor. There is an overlap, but it is not necessarily identical. At the moment, this lack of specific CSC markers is a major limitation in the identification of CSCs. Dr Ellis: Another issue is how do you measure CSCs in breast cancer or colon cancer or pancreatic cancer? We need more research to help us clearly identify a CSC population. Dr Mani: I believe CSC research will change our way of treating patients. Today, personalized cancer medicine focuses on the specific mutation in someone s tumor. Eventually, personalized treatment will consider CSC subpopulations as well. Dr Chang: Chemotherapy alone, combinations of chemotherapy, or targeted agents alone may not be the answer. They may not kill all the different clones of cells with different properties in the tumor, and they may not kill CSCs. You may need a combination approach that includes CSC-directed therapies. CONCLUSION Although the existence of CSCs was only proven a couple of decades ago, research suggests they may play fundamental roles in nearly all cancer processes. It is expected that oncologists will consider CSCs during the diagnostic workup and treatment decision-making. Rather than focusing solely on detecting and eliminating the majority population of rapidly dividing, terminally differentiated tumor cells, treatment efforts may also be directed at the subpopulation of CSCs and related signaling pathways that are the source of tumorigenesis, resistance, recurrence, and metastasis. 6 The identification of signaling pathways that regulate stemness of CSCs as well as CSC-specific markers that help isolate CSCs in tumors will provide important insights into the development of therapies that may provide greater specificity and decreased toxicity. 5,13 A multifaceted therapeutic approach that incorporates CSC-directed therapies into conventional treatment regimens may reduce the likelihood of treatment resistance, tumor aggressiveness, and tumor regrowth for patents with CSC-possessing cancers

11 GLOSSARY Cancer stem cell (CSC) Cancer cell that can self-renew, differentiate into progenitor and non-stem cancer cells, and generate serially transplantable heterogeneous tumors. The CSCs may underlie disease relapse and tumor metastasis. 3,4,17,47 CSC markers Protein expression, protein activity, and/or functional cellular signatures used to identify and enrich CSC populations. 3,17 Differentiation Process by which less specialized cells give rise to more specialized cells, resulting in a progressive decrease in self-renewal and multilineage developmental potential. 48 Epithelial-mesenchymal A normal tissue organization process that functions by converting transition (EMT) interconnected, anchored epithelial cells to disconnected, motile mesenchymal-like cells. Pathological activation of EMT in CSCs may support their stemness and facilitate invasion of lymphatic and circulatory systems to promote metastasis. 35,49,50 Microenvironment Cellular and acellular surroundings of a cell that affect its behavior. 51 The normal stem cell microenvironment, or stem cell niche, maintains stemness. 6 Alterations in the normal stem cell niche may produce a pathological niche supportive of CSC stemness, 14 treatment resistance, 11 and EMT. 25 Non-stem cancer cells Non-tumorigenic cells that make up the tumor bulk and have limited proliferative potential. 52 Plasticity Ability of differentiated cells to generate differentiated cells of the same or different lineage or to dedifferentiate to more primitive cell states in a reversible manner. In tumors, non-stem cancer cells may dedifferentiate to interconvert between differentiated and CSC-like states. 3 Quiescence State of temporary and reversible cellular non-proliferation. 53 Self-renewal Defining property of stem cells and CSCs in which parent cells divide to yield unaltered copies with the same self-renewal and differentiation potential. 47,54 Signaling pathway Network of interactions between cellular molecules that determine a route of signals that induce cellular responses. 55,56 Inter- or intracellular signaling molecules bind to receptor proteins, which subsequently alter or activate target proteins responsible for mediating downstream cellular function. 56,57 Dysregulation of signaling pathways underlies CSC maintenance. 2 Stem cells Unique, unspecialized cells capable of long-term self-renewal and differentiation. 58 However, most stem cells are quiescent, 3 and their differentiated progeny have limited plasticity. 3 Stem cells function in tissue/ organ development, injury repair, and tissue regeneration. 16,58 Stemness Defining characteristics of stem cells, including self-renewal to preservation of undifferentiated stem cells and production of progenies that can differentiate. 59 Treatment resistance An underlying cause of cancer progression and recurrence. Diverse mechanisms, such as anti-apoptosis protein overexpression, drug efflux proteins, DNA repair activation, low reactive oxygen species levels, quiescence, aberrant developmental signaling, and microenvironmental stimuli, contribute to the treatment-resistance property of CSCs

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