Biochimica et Biophysica Acta (BBA) - General Subjects
Developmental signaling pathways in cancer stem cells of solid tumors☆
Highlights
► Cancer may be viewed as a disease in which normal cellular homeostasis is disrupted. ► Developmental signaling pathways are often deregulated in cancer. ► Cancer stem cells have been identified and characterized in many solid tumors. ► Breast, brain and colon cancer stem cells are the most well-characterized. ► Hh, Notch and Wnt pathways play important roles in normal and cancer stem cells.
Introduction
Many tissues are composed of cells that are constantly turning over via cell proliferation, differentiation, and death. For normal tissue development and maintenance throughout life, a carefully regulated balance of these processes is required to maintain tissue homeostasis. During development and in adult tissues, cells are produced and localized in a defined spatial–temporal manner where their fate is specified by both internal and external signals. Errors that fail to be repaired can dramatically shift the homeostatic balance leading to the initiation, expansion and progression of an abnormal mass of cells that no longer respond to normal signaling cues (see Review [1]). Understanding the mechanisms of normal tissue development and homeostatic control will therefore provide important clues as to how to rein in the ‘uncontrolled behavior’ of cancer cells.
At the time of detection tumors generally display extensive cellular heterogeneity, containing cells that differ morphologically, functionally and molecularly. Two models have been proposed to explain intra-tumoral heterogeneity. The conventional stochastic or clonal evolution model postulates that all cells within a tumor have the potential to give rise to new tumors and that distinct clones adapt and evolve to produce genetically and phenotypically distinct cells. On the other hand, the more recently proposed cancer stem cell model postulates that there exists a subset of tumor cells that display stem cell-like properties, originally discovered in leukemia [2], [3] and reviewed in [4], [5]. These cells are termed cancer stem cells (CSCs), tumor-initiating cells (TICs) or tumor-propagating cells (TPCs). CSCs are defined by their capacity to self-renew, differentiate and initiate tumor growth in vivo that recapitulates features of the original tumor. A level of hierarchy is maintained, and similar to normal stem cells, CSCs give rise to tumor progeny that are more differentiated and do not have the ability to regenerate a tumor. However, the clonal evolution and CSC models are not necessarily mutually exclusive. For instance, it is has been demonstrated in acute lymphocytic leukemia (ALL) that patient samples contain multiple genetically distinct subclones of cancer-initiating cells [6] indicating that the CSC-containing sub-fractions within tumors undergo clonal evolution. Thus it is likely that tumor heterogeneity can be explained using concepts from both models. Analysis of the genetic landscape of tumors, and more specifically the genetics of the CSC population within and across tumors, is currently an area of intensive investigation.
A CSC population was first identified in acute myeloid leukemia (AML) where a subset of cancer cells showed serial transplantation ability [3]. CSCs from solid tumors were more recently identified first from breast cancers [7] and then from several others including the brain [8], colon [9], [10], [11], head and neck [12], pancreatic [13], [14], melanoma [15], mesenchymal [16], hepatic [17], lung [18], prostate [19] and ovarian [20] cancers. However, not all cancers adhere to the hierarchical model [21], [22], [23] and there has been considerable debate about the accuracy of the CSC hypothesis, particularly in studies using human solid tumors and potentially inaccurate xenograft transplantation assays [24]. More recently, studies in mouse models of cancer have provided convincing evidence to support the CSC hypothesis in several cancer types. In these studies, lineage-tracing was used to demonstrate that in the intestine, brain, and skin specific cell subsets marked by stem cell-specific expression of fluorescent proteins act as cancer stem cells, clonally giving rise to malignancies and producing non-malignant, differentiated progeny as well as, in one case, being resistant to chemotherapy [25], [26], [27].
In studies of human cancers, CSCs are typically identified and isolated based on biochemical properties such as Hoechst dye efflux or aldehyde dehydrogenase-1 (ALDH1) activity, or by specific cell surface marker expression. In some cancers, such as AML, robust markers have been identified, whereas markers for CSCs in several solid tumors still require further refinement in order to be applied to cancer treatment. Typically, candidate CSC markers are validated using in vivo limiting dilution assays (LDAs) to determine the frequency of cancer cells capable of initiating tumor growth. Bulk tumor or tumor cell fractions isolated by cell-sorting techniques are injected into immunocompromised mice at limiting doses and the frequency at which each population of cells initiates tumor growth is calculated. Cell surface or biochemical markers that successfully enrich for a CSC population will give rise to tumors at low doses that recapitulate the original tumor. CSCs, but not non-CSCs, can be serially isolated and propagated for several generations of mice. Moreover, in vitro sphere-formation assays have been increasingly utilized to isolate and study CSCs, however, they should be used to complement rather than replace the gold standard of in vivo LDA experiments.
Since CSCs possess properties similar to normal stem cells, it has been suggested that the initiating event for cancers occurs in the long-lived normal stem cell compartment [2]. In tissues with high turnover rates, such as blood, skin, and intestinal epithelium, the stem cells may be the only cells sufficiently long-lived to sustain the multiple oncogenic hits that ultimately lead to transformation. However, alternative mechanisms have been proposed in which more differentiated cells may become CSCs. In this case, an early oncogenic event would be required to activate a stem cell-like program that would allow for survival and self-renewal abilities. Indeed, fully differentiated cells such as fibroblasts can be ‘de-differentiated’ or ‘reprogrammed’ when stem cell genes (Oct4, Sox2, Klf4 and c-Myc) are over-expressed [28]. This finding supports the notion that any cell along a lineage restricting path from a stem cell to a differentiated cell could be a target if the right genetic programs become activated. Moreover, a block in differentiation events may also result in a prolonged life span of these normally short-lived differentiated cells that could favor later transforming events. One example of this is differentiation-inducing transcription factor CCAAT enhancer binding protein alpha (C/EBPα), a critical regulator of tissue-specific gene expression and proliferation arrest [29]. Loss of function of C/EBPα occurs in acute leukemias, and has also been implicated in non-small cell lung cancer, and squamous cell carcinomas of the skin and head and neck [29]. Alternatively, pre-disposing “hits” may occur in the stem cell compartment, with the final transforming event occurring in a more differentiated downstream cell. For example, in chronic myeloid leukemia (CML), the initial event (BCR–ABL fusion) occurs in the hematopoietic stem cell compartment, but upon transition from chronic phase to blast crisis, an additional event (activation of Wnt signaling) occurs in the granulocyte–macrophage progenitor, which then acquires self-renewal ability and takes on the role of CSC in this disease [30]. Regardless of the cell of origin, the CSCs that are isolated from tumors display self-renewal properties at both the molecular and functional level. CSCs express self renewing genes and exhibit activation of the developmental signaling pathways that are essential in regulating stem cell activity. Heterogeneity among and between different cancers presents challenges to specifically defining CSCs genetically, molecularly, phenotypically and functionally. Clearly, CSCs are able to self renew and initiate tumor growth; however, the sequence of events by which a CSC acquired these features, as well as how they maintain these features, is likely very different among tumors. For instance, some CSCs may be more dependent on the Wnt signaling pathway compared to others that may utilize the Hedgehog pathway. Understanding the underpinnings of what defines a CSC population within a specific tumor of interest is essential if CSCs are to be targeted for therapies. In the following sections, it will become evident that despite the shared property of self renewal, CSCs do NOT necessarily share the same mechanisms of self renewal and this represents a great challenge for being able to target specific pathways. Signaling pathways that have been discovered to play a role will be discussed.
During embryogenesis, a myriad of signaling pathways are involved in the development of distinct tissues. Identified stem cell related genes include BMI1, OCT4, SOX2, and NANOG and the self renewal properties of both embryonic and adult stem cells are regulated by key developmental signaling pathways such as Notch, Wnt and Hedgehog (Hh). Adult stem cells usually reside in a niche that is under strict control and highly enriched for the activity of these signaling pathways. Interestingly, the genes involved in these signaling pathways are found to be frequently mutated or aberrantly activated in cancers and it is the deregulation of these pathways that has been linked to CSC function and maintenance.
In summary, if CSCs are the driving force behind tumor maintenance and growth then understanding the molecular mechanisms regulating CSCs is essential. Evidence suggests that CSC function depends on ligand-receptor mediated signaling pathways. The Notch, Wnt and Hh signaling pathways (Fig. 1) are conserved in regulating normal stem cell activity and their deregulation is linked to oncogenesis. This review will focus on the role of the Notch, Wnt and Hh signaling pathways in CSCs from the brain, breast and colon cancers, as these have been the most extensively studied.
Section snippets
Notch
The Notch signaling pathway is evolutionarily conserved and plays many roles such as influencing stem cell fate, differentiation and cell cycle progression. Notch signaling can be either inhibitory or inductive which is highly context dependent [31]. For instance, during brain development, Notch serves as a gate keeper for cell fate where gliogenesis can only occur when Notch signaling specifically represses the neuronal pathway in the progenitor cells. In Drosophila, Notch signaling induces
Isolation of normal neural stem cells
Neural stem cells (NSCs) are located in the subventricular zone (SVZ), the hippocampus and the subgranule zone (SGZ) of the adult brain [39], [40], [41]. They were initially isolated from mice using an in vitro assay in which they could form single cell-derived “neurospheres” that functionally engrafted into mouse brains and had the potential to differentiate into all three brain lineages (astrocyte, oligodendrocyte and neuron) [42], [43]. In serum-free conditions normal NSCs display activation
Concluding remarks
While there has been much controversy surrounding the CSC hypothesis, it is clear that many tumors are in fact caricatures of the normal tissues in which they arise. This is supported by the similarities in phenotypes between normal stem cells and CSCs, and by the role of deregulation of pathways that are normally essential for embryonic development and maintenance of tissue homeostasis in cancer initiation and progression, as described above. While the Notch, Hh and Wnt pathways clearly play
Acknowledgements
This research was funded in part by the Ontario Ministry of Health and Long Term Care. The views expressed do not necessarily reflect those of the OMOHLTC. In addition, this work was supported by the Ontario Cancer Institute/Princess Margaret Hospital, Campbell Family Institute for Cancer Research and the Ontario Institute for Cancer Research.
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This article is part of a Special Issue entitled Biochemistry of Stem Cells.