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Keywords:

  • Colorectal cancer;
  • Stem cells;
  • Microenvironment;
  • Epithelial to mesenchymal transition;
  • Metastasis

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

Colorectal cancer (CRC) is one of the most commonly diagnosed and lethal cancers worldwide. It is a multistep process that requires the accumulation of genetic/epigenetic aberrations. There are several issues concerning colorectal carcinogenesis that remain unanswered, such as the cell of origin and the type of cells that propagate the tumor after its initiation. There are two models of carcinogenesis: the stochastic and the cancer stem cell (CSC) model. According to the stochastic model, any kind of cell is capable of initiating and promoting cancer development, whereas the CSC model suggests that tumors are hierarchically organized and only CSCs possess cancer-promoting potential. Moreover, various molecular pathways, such as Wingless/Int (Wnt) and Notch, as well as the complex crosstalk network between microenvironment and CSCs, are involved in CRC. Identification of CSCs remains controversial due to the lack of widely accepted specific molecular markers. CSCs are responsible for tumor relapse, because conventional drugs fail to eliminate the CSC reservoir. Therefore, the design of CSC-targeted interventions is a rational target, which will enhance responsiveness to traditional therapeutic strategies and reduce local recurrence and metastasis. This review discusses the implications of the newly introduced CSC model in CRC, the markers used up to now for CSC identification, and its potential implications in the design of novel therapeutic approaches. STEM CELLS 2012;30:363–371


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide with more than 1.2 million new cancer cases and 600,000 deaths estimated to have occurred in 2008. It is the third most commonly diagnosed cancer in males and the second in females [1]. CRC is presented as a multistep genetic disorder characterized by specific mutations in signal transduction pathways. The development and progression from adenoma to cancer and metastatic disease require the simultaneous failure of protective mechanisms, including adenomatous polyposis coli (APC), p53, and transforming growth factor β (TGF-β), as well as the induction of oncogenic pathways, such as Ras [2, 3]. Traditional models of tumorigenesis suggest that every cell within the tumor population is capable of tumor initiation and propagation. The newly discussed cancer stem cell (CSC) model, however, proposes that only a small fraction of cells possesses tumor propagation abilities [4]. This hypothesis raises questions regarding the efficiency of current diagnostic and therapeutic measures, suggesting that CSCs are a rational target for the development of robust diagnostic, therapeutic, and follow-up strategies.

NORMAL COLON STEM CELLS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

The large intestine is divided into four distinct anatomical layers. The inner luminal lining is a single layer of epithelial cells folded into finger-like invaginations, which are embedded in the submucosal connective tissue to form the functional unit of the intestine, the crypt of Lieberkuhn [5]. Each crypt contains around 2,000 cells and comprises three main terminally differentiated cell lineages (enterocytes, goblet cells, and endocrine cells) that reside in the top-third of the crypt, and possibly Paneth-like cells [6, 7]. These cells are under a never-ending replacement process, as they constantly shed into the lumen when they become senescent [8, 9]. The replacement rate of intestinal epithelium (5 days) is regulated by stem cells (SCs) and is under microenvironmental influence [5, 7]. Intestinal SCs are undifferentiated, multipotent, and self-renewable/maintained cells involved in tissue homeostasis and repair, located at the crypt base [10]. These cells divide mostly asymmetrically and give rise to two different daughter cells, with one being identical to the original cell, while the other has the potential to differentiate (progenitor). Progenitors migrate to the top of the crypt and reproduce the fully differentiated intestinal repertoire (Fig. 1). Symmetric divisions may occur during injury, disease, or cancer [10–12]. Colon shares this SC-based hierarchical organization with many other tissues/systems that are under constant renewal, including the hematopoietic system and the skin [11]. Originally, the Unitarian theory proposed the monoclonal nature of intestinal crypts [10]. More recent evidence, however, implies that crypts are probably polyclonal at birth and subsequently shift toward monoclonality by following a pattern of neutral drift [7, 13]. An intestinal crypt contains approximately 16 SCs and harbors two distinct pools of putative SCs. The one pool is located at the crypt base and is characterized by the expression of leucine-rich repeat containing G protein-coupled receptor 5 (Lgr-5) and the other pool resides at +4 position and consists of B lymphoma Moloney murine leukemia virus (Mo-MLV) insertion region 1 homolog (Bmi-1) and telomerase reverse transcriptase (Tert) expressing cells [13, 14]. The contribution of each type of cell to the maintenance of the SC pool is under debate. One possible suggestion is the existence of a pool of equally contributing cells where each cell's behavior is defined by its entourage and in terms of replacement they follow a pattern of neutral drift [13]. An alternative approach is that Lgr-5+ SCs comprise the active population of the crypt, whereas Bmi-1+ or Tert+ cells are quiescent SCs that represent a reserve pool of SCs with the ability to replace Lgr-5+ cells in case of loss or injury [7, 14]. Moreover, it has been proposed that Bmi-1+ cells may not have an impact on Lgr-5+ SCs, as Bmi-1 knockout mice show normal crypt morphology and have a normal intestinal epithelium [15].

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Figure 1. Hierarchical organization of a normal colonic crypt—comparison between the stochastic and the CSC model. Colonic crypts are hierarchically organized. SCs divide into progenitor cells that migrate at top and produce terminally differentiated cells. In the stochastic model, all cells possess tumor initiation potential. In the CSC model, only CSCs are capable of tumor propagation and metastasis. Microenvironmental crosstalk, epigenetic, and genetic aberrations (yellow lightning bolt) may lead to clonal evolution (dark purple cells). Circumscribed cells represent the mutated types of their normal homologs (white polygons: A, asymmetrical division; S, symmetrical division). Abbreviations: BMP, bone morphogenetic protein; CBCC, crypt base columnar cell; CSC, cancer stem cell; D, differentiated cell; P, progenitor cell; SC, stem cell; WNT, Wingless/Int.

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SC NICHE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

The functional importance of the intestinal microenvironment (niche) in the advanced control of SC life cycle is widely accepted. The niche consists of cellular and extracellular components that ensure the optimal conditions for SC maintenance through the secretion of various cytokines, growth factors, and direct interactions [10, 16]. Interestingly, intestinal SCs may also be affected by components in the crypt lumen, derived from epithelial cells or from bacteria. Intestinal subepithelial myofibroblasts are key regulators of SC self-renewal and differentiation, mediate the crosstalk between epithelial and mesenchymal cells, and secrete a wide range of morphogenetic factors [7]. Epithelial–mesenchymal interactions regulate the normal intestinal architecture and additionally define the balance between proliferation and differentiation [10, 16]. Wingless/Int (Wnt), Hedgehog, bone morphogenetic protein (BMP), Notch, and platelet-derived growth factor pathways are involved in these interactions [7].

IDENTIFICATION OF NORMAL INTESTINAL SCs

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

Identification and isolation of SCs remain an issue of debate [8] due to the lack of specific molecular markers along with unsolved technical issues, forcing the use of a functional definition based on the attributes of self-renewal and multipotency. Consequently, only assumptions can be made regarding their exact number and position [17]. Several studies have been carried out, mostly on small intestine, which used DNA labeling techniques to exploit the comparatively slower cycling rate of SCs [18] and led to the formulation of two different models regarding their position [5]. The +4 position model postulates that SCs are located at the +4 position of the small-intestine crypt just above the Paneth cells, which occupy the bottom. These cells express the markers Bmi-1 and Tert. SCs divide into progenitor cells that migrate to the top of the crypt and differentiate, while Paneth cells follow the exact opposite direction [7, 16]. Alternatively, the SC zone model was proposed after discovering the presence of small immature cycling cells at the crypt base between Paneth cells. These cells were named crypt base columnar cells and express the Wnt target gene Lgr-5 [19]. Recently, many molecules, mostly located on the cell surface, have been proposed as putative stemness markers, at the same time allowing isolation by fluorescence-activated cell sorting [10]. In summary,the molecules involved in critical stages of proliferation and differentiation are Musashi-1 (Msi-1), CD29, Bmi-1, Lgr-5, aldehyde dehydrogenase 1 (ALDH-1), Tert, and achaete scute-like 2 [5, 7, 9, 10, 14, 15]. For a more detailed presentation of the currently used markers, the reader is referred to Table 1.

Table 1. Normal colon stem cells' markers
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COLORECTAL CARCINOGENESIS—THE CSC MODEL

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

Colorectal carcinogenesis results from a series of genetic/epigenetic alterations and interactions with microenvironmental and germ-line factors that transform the normal colonic mucosa into an aberrant phenotype [2, 3]. The questions which is the cell of origin and which cells propagate the tumor after initiation remain elusive despite extensive in vitro and in vivo experiments [7, 8]. The cell of origin is the cell that acquires tumorigenic mutations and becomes the first tumor cell. A cell of origin is different from the cells responsible for tumor propagation after its inception [8]. Every normal cell (SC, progenitor, or differentiated) can accumulate mutations and become the cell of origin [8, 10, 24]. Following its malignant transformation, it is possible to follow one of the two proposed models for the explanation of carcinogenesis and progression, the stochastic or the CSC model (Fig. 1). Even if a normal SC becomes the cell of origin, it can form a tumor according to the features of the stochastic or the CSC model [8]. The stochastic model suggests that every cell within a tumor is capable of both initiation and propagation. After transformation, each cell can acquire further mutations. The heterogeneity within a tumor can be explained by the aggregation of random genetic aberrations as well as the divergent microenvironmental influence [8, 25]. Concomitantly, a diverse population of subclones with different properties is generated. Only selected clones can migrate and form metastases. The migrating clones can further accumulate mutations, resulting in a divergent metastatic tumor from the parent lesion [11]. The newly introduced CSC model, on other hand, states that only a small portion of cells within a tumor is endowed with tumor propagation potential and thus named CSCs, whereas all other cells are not [26]. According to the CSC model, similar to the normal tissue, cancers are also hierarchically organized. Tumor heterogeneity results from the production by the multipotent CSCs (high proliferative) of a wide variety of progenitor (medium proliferative) and differentiated (nonproliferative) cells [11, 27]. In this model, only CSCs can migrate and as a result the metastatic tissue resembles the pattern of the original lesion. However, individual cell responses to microenvironmental stimuli, epigenetic modifications, and additional genetic aberrations may be observed within a cancerous tissue, which in turn may lead to clonal evolution and gain or loss of CSC attributes. A new and improved CSC due to clonal evolution may arise, which will now drive tumor growth and metastasis instead of the initial CSC [8, 28]. CSCs are characterized by self-renewal, multipotency, limitless proliferation potential, angiogenic, and immune evasion features [24]. Intriguingly, CSCs are relatively highly resistant to traditional tumor therapeutic measures and thus responsible for tumor relapse due to the expression of DNA repair mechanisms, detoxifying enzymes, and drug transporters [29]. Most likely, CSCs are derived from normal SCs due to their extended life span that alter their behavior as a result of genetic and epigenetic changes. It has also been discussed that progenitor, differentiated, or even cells from outside the tumor, for example, bone marrow-derived cells, might also serve as CSCs' ancestors [8, 10, 11, 28, 30]. Concomitantly, through symmetric (CSC overproduction) or asymmetric (heterogeneity) divisions, the entire crypt will be colonized from the mutated SCs and their descendants. Future alterations may lead to a more aggressive and metastatic phenotype [5, 31]. Throughout CRC progression, the number of CSCs remains approximately 1% of the number of total cells. However, their frequency appears to be highly variable [6, 28]. The CSC model modifies the classic Fearon and Vogelstein model, which is characterized by step-by-step genetic modifications of the adenoma to carcinoma sequence, by placing the normal SC as the primary candidate for being the cell of origin, by underlying the crucial importance of microenvironmental signals, and by explaining tumor heterogeneity within the context of a clonally evolved CSC model [7, 10].

SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

The intestinal homeostasis is under the surveillance of a complex crosstalk network of evolutionary conserved pathways, such as Wnt, Notch, and Hedgehog, which control the balance between proliferation, differentiation, migration, and renewal. The Wnt pathway is responsible for endoderm formation and exerts fundamental role in crypt development, maintenance, and proliferation, as it is depicted by the failure of Wnt knockout mice to develop colonic crypts [8, 18, 32]. C-myc, cyclin D1, and Lgr-5 are included among its various targets. A Wnt gradient loss of expression from the base to the top is closely related with differentiation and expression of the ephrin family proteins, which regulate positioning [5]. Mutations in the APC gene (80% in sporadic cancer), β-catenin, or the regulatory proteins in the Wnt pathway result in constant activation [18]. This may lead to uncontrolled proliferation, a shift from asymmetrical to symmetrical divisions, and augmented survival. Wnt signaling is also involved in the process of epithelial to mesenchymal transition (EMT) and invasion [7, 8]. Notch may drive tumorigenesis, because it potentiates proliferation and inhibits differentiation [10]. BMP and TGF-β belong to a family of ligands whose receptors interact with the intracellular cascade of the Smad proteins [33]. BMPs are mostly expressed by stromal cells, have opposing actions to Wnt, and are mostly active at the top of the crypt. In the crypt bottom, they are also produced, but they are kept inactive by binding to specific inhibitors [7, 16]. TGF-β is an inhibitor of intestinal epithelial cell proliferation and inducer of apoptosis [3, 31, 34]. The Hedgehog proteins carry signals between stromal and epithelial cells. Although it seems that Hedgehog acts as a Wnt suppressor, probably through BMP, and is also expressed by +4 position stem-like cells, its role remains elusive [7]. Finally, in colorectal carcinogenesis, activation of the phosphoinositide 3-kinase (PI3K) pathway results in rapid and excessive proliferation of intestinal SCs [12].

COLORECTAL CSC NICHE

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

Like the normal SCs, CSCs reside in a qualified microenvironment that has gained tumor-promoting traits [35]. A reciprocal interplay between SCs and their niche is behind malignant transformation. Genetic or epigenetic aberrations in the SCs compartment may lead to alteration of the niche and vice versa [28, 36]. This tumorigenic niche is composed of transformed myofibroblasts, recruited myeloid cells, other cell types, and extracellular components, which produce various growth factors and cytokines, including hepatocyte growth factor (HGF), tumor necrosis factor α (TNF-α), and interleukin (IL)-6, which promote dedifferentiation, carcinogenesis, and invasiveness [7, 36]. Although Wnt activating APC mutations are common in CRC, tumors often present with a heterogeneous Wnt activity, indicating an alternative regulation. Myofibroblast-derived HGF is probably a major enhancer of Wnt activity [7, 36]. Nuclear β-catenin localization, which indicates Wnt-triggering and cancer-stemness, is predominantly observed in the invasive regions of colorectal carcinomas [37]. Experimental data revealed that mice carrying an APC mutation combined with Smad4 loss, and thus unresponsive to differentiation-inducing microenvironmental signals, showed increased susceptibility in the formation of highly invasive tumors combined with extensive stromal proliferation [38]. In another study, it was shown that inactivation of stromal BMP may also lead to extensive proliferation of both the stroma and the overlying epithelium and eventually adenoma formation. Wnt activity was increased in epithelial cells and was under the manipulation of other stromal pathways, such as Forkhead Box L1 (FOXL1) and phosphatase and tensin homolog (PTEN) [12]. In humans, evidence that supports this relationship is derived from observations in patients with inflammatory bowel disease, histopathological data of stromal immune cells, and the beneficial role of nonsteroidal anti-inflammatory drugs in CRC. The chronic inflammatory milieu, composed of infiltrating macrophages, lymphocytes, and stromal cells, which produce IL-1, IL-6, TNF-α, and vascular endothelial growth factor, disturbs epithelial homeostasis and the SC reservoir [6]. It has been postulated that a normal intestinal niche can prevent tumor growth, even if transformed SCs are present [39].

EMT AND METASTASIS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

Metastasis is responsible for approximately 90% of cancer-associated mortality and can be divided into translocation and colonization basically [40]. As the CSC population represents the only cells that propagate tumors, it can be extrapolated that these cells are responsible for metastasis formation [41, 42]. It is therefore a prerequisite that these cells must be able to detach from the primary tumor, invade, access, and survive at the circulation, disseminate at distant sites, transmigrate across the endothelial lining of the target tissue, and form secondary tumors (Fig. 2) [43]. A crucial step in the metastatic process is EMT. In this event, the simultaneous downregulation of the epithelial phenotype, along with enabling of fibroblast-like traits, enhances motility, invasiveness, and resistance to apoptosis [40, 44]. Cells that undergo EMT are also assumed to gain a stem-like phenotype [45, 46]. This process seems to be regulated by pathways that are linked with SCs, such as Wnt and TGF-β [28]. Studies in CRCs revealed a mixed epithelial (tumor center) and mesenchymal (invasive front, EMT/stemness) phenotype in both the primary tumor and the metastasis [37, 45]. Removal of contextual signaling may reverse this process [36, 47]. The proposed plasticity of CSCs brings new insights in the field, suggesting that not only CSCs produce more differentiated descendants but also non-CSCs can be converted to CSCs through EMT. The reprogramming is mediated by microenvironmental signals [7, 36, 47, 48]. To enlighten the microenvironmental-driven plasticity of CSCs, two models have been proposed. The intrinsic model suggests that certain cell population possesses CSC traits from tumor initiation. Meanwhile, extrinsic signals, derived from recruited active stromal cells, force cells to undergo EMT and to adopt CSCs aspects [40, 48]. Therefore, the existence of a circulating tumor cell (CTC) with stem-like properties is speculated [40, 49]. Data from breast, lung, and colon cancer patients indicate that not only current CTC detection techniques tend to underestimate the population of CTCs with stem-like and EMT traits but also that the presence of stemness markers in the peripheral blood of cancer patients is associated with worse prognosis and recurrence [50–52]. It seems that through EMT, CTCs adopt a more aggressive and SC-like phenotype. However, understanding the exact relation between EMT, CSCs, and CTCs will provide additional tools for the diagnosis and therapy of cancer patients.

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Figure 2. The stem cell model of metastasis. The primary tumor releases in circulation CSCs that have underwent EMT and possibly clonally evolved (dark purple and dark purple ovular flattened cells) due to accumulating genetic and epigenetic modifications (yellow lightning bolt). These cells travel to distant organs, where they formulate metastases. The secondary organs may provide a niche for the tumor cells. Steps 1–5 (white polygons) indicate the consequent stages required for the transition from local to metastatic disease. Abbreviations: CSC, cancer stem cell; D, differentiated cell; EMT, epithelial to mesenchymal transition; P, progenitor cell.

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IDENTIFICATION OF COLORECTAL CSCs

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

Although CSCs have been implicated in colon carcinogenesis, their existence had not been experimentally demonstrated until recently. However, due to the complexity of their biology and unsolved technical issues, an unequivocally approved identification and isolation strategy is still a matter of debate [9, 31]. Experimental studies exploit basic, but not exclusive, features of CSCs, such as the long-term retention of DNA label, morphological traits, epigenetic modifications, or differential gene expression [53–55]. Most recently used and prominent techniques, such as immunohistochemistry and cell sorting isolation, are based on the expression of possible stemness markers [56, 57]. The tumorigenic potential of isolated cells is often evaluated by their ability to reproduce the full heterogeneous repertoire of the parent tumor when xenotransplanted in immunodeficient mice or through colony forming assays [28]. Certain speculations arise, however, regarding the reliability of these assays. The environment of the host organism does not resemble exactly the original conditions and, additionally, cytokines and growth factors from different species may show limited affinity for their related receptors [12, 26, 28]. The site of transplantation may also influence the experimental outcome, as an orthotopic assay for colon CSCs has not been described yet. Further issues may occur due to the alteration of certain markers caused by culturing and sorting conditions [12, 26, 28]. The presence of residual immune cells in the recipient organism and an altered vascular environment may influence the tumorigenic potential of injected cells [28]. Several molecules have been proposed as CSC markers including CD133, CD44, CD24, CD166, Lgr-5, and ALDH-1 (Table 2). CD133, a pentaspan transmembrane glycoprotein implicated in the organization of plasma membrane [9], was first reported as a putative colorectal CSC marker in the studies of O'Brien et al. and Ricci-Vitiani et al. by injecting CD133 positive and negative (sorted) cells in immunodeficient mice. Only a small subset of CD133+ cells was capable of initiating tumor growth, while negative cells were not. In both studies, CD133 expression was observed in normal colon, although at lower numbers, suggesting a possible relation between normal and cancer SCs [58, 59]. Following this breakthrough, several other groups investigated the expression patterns of CD133 in CRC cell lines or biopsy specimens. CD133 expression was independently correlated with SC potential, worse prognosis, and low survival. Moreover, its combined expression with other putative CSC markers was also evaluated [20, 60-67]. It seems, however, that most CD133 antibodies target glycosylation-dependent epitopes [20], whose presence is related to the differentiation stage of the cell. Recent experimental data from colon and glioblastoma cells suggest that the differential glycosylation of specific epitopes may mask the presence of CD133 on cells previously characterized as negative [68, 69]. Nevertheless, data revealing that CD133 is not restricted to normal or cancer SCs raised questions regarding its efficiency as a colorectal CSC marker. CD133 was ubiquitously expressed in differentiated colonic epithelium and in tumor cells. Both CD133+/− isolated cells were able to form tumors when injected in mice [70]. The cell adhesion molecule CD44 was proposed as an alternative marker. CD44+ cells exhibited CSC properties, as a single cell could form a sphere and a xenograft tumor that resembled the original lesion [71]. Its expression in CRC has been negatively associated with the depth of invasion, lymph node infiltration, prognosis, and survival [72]. Albeit CD133 and CD44 seem to be promising markers, as cells expressing this protein display tumorigenic potential in vitro and in vivo, a combination of markers is likely to be more suitable for detecting colorectal CSCs [73]. The high heterogeneity of human cancers forces the thorough investigation of each individual subpopulation [55]. Additional markers include CD166, epithelial cell adhesion molecule, ALDH-1, CD29, CD24, CD26, Msi-1, Lgr-5, and Wnt activity/β-catenin. The presence of these molecules was associated with stemness characteristics both in vitro and in vivo, breeding tumors that resemble the original lesion and displaying increased clonogenic ability, tumorigenicity, and multilineage potential. Associations were also made with tumor stage, differentiation, invasiveness, metastasis formation, prognosis, and survival. These markers were also used for further enrichment of isolated CSCs to enhance their tumorigenic ability [18, 21–23, 36, 74-90]. The pluripotency genes Oct-4, Sox-2, Nanog, Lin-28, Klf-4, and c-myc are regarded as promising surrogate markers. These genes seem to facilitate a shift toward an undifferentiated state [45, 91]. Their expression has been associated with poor prognosis, relapse, and distant recurrence, as well as with resistance to conventional chemotherapy/radiotherapy [91–93]. Interestingly, the CD133 negative cell line NANK displayed tumor initiating abilities when injected in mice. Despite lacking the expression of conventional CSC markers CD133 and CD44 as well as the self-renewal genes Oct-4 and Nanog, NANK cells expressed the developmental markers CDX2 and Bmi-1 [94].

Table 2. Colorectal cancer stem cells' markers
  1. Abbreviations: ALCAM, activated leukocyte cell adhesion molecule; ALDH-1, aldehyde dehydrogenase-1; APC, adenomatous polyposis coli; Bmi-1, B lymphoma Mo-MLV insertion region 1 homolog; CRC, colorectal cancer; EpCAM, epithelial cell adhesion molecule; Lgr-5, leucine-rich repeat containing G protein-coupled receptor 5; Msi-1, musashi-1; SC, stem cell.

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THERAPEUTIC CHALLENGES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

According to the CSC model, tumor growth is sustained by a small population of cells that current therapeutic measures fail to eradicate. Although antitumor strategies eliminate the vast majority of cancer cells, leading to a reduction in tumor size and disease remission, they fail to prevent relapse by leaving intact the CSC reservoir. On the contrary, although a CSC-targeted therapy will not cause tumor-size shrinkage in short term, it will prevent future regrowth and metastasis [11, 95]. Experimental data support the notion that CSCs are responsible for resistance to chemoradiotherapy and disease relapse [51, 89, 93, 96]. Resistance is exhibited either through a shift from active state to quiescence or a wide spectrum of protective mechanisms (DNA damage repair, altered cell-cycle checkpoint control, malfunction of apoptosis, drug transporters, and detoxifying enzymes) [29, 40, 97]. The proposed plasticity of CSCs complicates therapeutic approaches because not only CSCs and cancer cells with CSCs traits that occur through EMT are more resistant to conventional treatment but also a strategy that targets only CSCs will fail to efficiently cure CRC [47]. EMT and CSC plasticity participate in the acquisition of both de novo and acquired drug resistance [44]. It is therefore essential to renovate our therapeutic arsenal by designing new curative methods that specifically target CSCs and also solve issues that arise from CSC flexibility by eliminating at the same time the non-CSC population and intervening in the process of EMT [44, 47]. Successful therapeutic interventions should dispatch all proliferating cells, eradicate the CSC reservoir, and then eliminate disseminated/circulating cells including those that may have become dormant [26, 95]. Several issues should be taken into account during the design of CSC-directed interventions. Possible adverse reactions may occur from the impact on normal SCs, which bear resemblance to their tumor relatives. This potential toxic effect can be minimized by targeting molecules or pathways that are preferentially active in CSCs [57]. Moreover, the development of resistance along with the heterogeneous nature of tumors points to the combination of agents [97]. Monoclonal antibodies could be directed against cell surface molecules, such as CD133, CD44, or even drug transporters. Inhibition of these molecules may result in reduction of tumor size, metastatic potential, and resistance to chemoradiotherapy [24, 57]. Another rational target includes blockage of various self-renewal pathways, including Wnt, Notch, PTEN, and Hedgehog [4]. Small molecular inhibitors of Wnt and γ-secretase inhibitors of Notch have been suggested as novel agents against CRC [10]. An alternative approach is the induction of differentiation and the disruption of EMT. Differentiation therapy forces cells to shift into a mature phenotype, lose their self-renewal abilities, and therefore become vulnerable to conventional treatment. Targeting EMT will contribute to the depletion of new CSCs that arise as products of EMT and in the blockage of drug resistance. This strategy includes: (a) alterations in signaling cascades, for example, BMP4 or PI3K, (b) application of microRNAs that alter gene expression profiles, and (c) epigenetic therapy [24, 44, 95]. BMP promoted differentiation and apoptosis in colorectal CSCs through modulations in the Wnt signaling cascade. Administration of oxaliplatin and 5-flouroucil, which enhance BMP antitumor action, resulted in the remission of colorectal CSC-derived xenograft tumors [10]. Reversing chemoresistance and radioresistance represents a promising proposal. This can be achieved through interference with a plethora of cellular components, including inactivation of drug transporters and DNA checkpoint kinases, depletion of reactive oxygen species scavengers, and inhibition of signal transduction pathways [97]. Recently, inhibition of the IL-4 pathway with an anti-IL-4 antibody or an IL-4 receptor antagonist in CD133+ colorectal CSCs augmented the antitumor effects of conventional chemotherapeutics. IL-4 functions in an autocrine manner, promoting antiapoptotic pathways [98]. The “malignant” microenvironment has been proven to be essential for the maintenance and development of CSCs. Interruption of the crosstalk network between the elements of the niche and CSCs will drive the latter in restriction of growth and loss of their metastatic capacity [4, 57]. Evidence from other malignancies supports the notion that tumor initiation and propagation may be mediated, at least in part, through evasion of host immune responses [57]. The application of the bisphonate zolendronate on colorectal CSCs promoted an efficient immune T-cell response [10].

CONCLUSIONS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES

The understanding of the molecular mechanisms implicated in the biology of CSCs as well as the identification of specific markers, which will permit efficient isolation, may aid efforts toward targeted treatment, more efficient diagnosis, and follow-up of CRC patients. CSCs are rational targets for the design of interventions that will enhance responsiveness to traditional therapeutic strategies and contribute in the reduction of local recurrence and metastasis.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. NORMAL COLON STEM CELLS
  5. SC NICHE
  6. IDENTIFICATION OF NORMAL INTESTINAL SCs
  7. COLORECTAL CARCINOGENESIS—THE CSC MODEL
  8. SIGNALING PATHWAYS: NORMAL COLON VERSUS CRC
  9. COLORECTAL CSC NICHE
  10. EMT AND METASTASIS
  11. IDENTIFICATION OF COLORECTAL CSCs
  12. THERAPEUTIC CHALLENGES
  13. CONCLUSIONS
  14. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  15. REFERENCES