Cancer Stem Cells and Biology
The cancer stem cell (CSCs) are distinguished as a small population of tumor cells which are able to form phenotypically diverse tumors, as wells as self-renew and differentiate. They are described as belonging to a group of tumor initiating cells (TICs) which may or may not possess stem-like characteristics, but debate remains as to how large a proportion of TICs are indeed stem-like. Additionally, it is not clear whether or not the plasticity of tumor cells allows any cell to become stem-like and gain the capability to recapitulate heterogeneous tumors. The role of CSCs in tumor formation was first identified by Bonnet and Dick in the late 90s .
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Cancer Stem Cells (CSCs) are either formed upon carcinogenesis of somatic cells or stem cells, or they are activated after a period of dormancy (1). These CSCs then asymmetrically divide resulting in a phenotypically diverse tumor consisting of both CSCs and non-stem-like cells (2). Left untreated, the tumor will continue to grow and invade the surrounding tissue, and CSCs undergoing EMT may break off from the original tumor and travel to distant organs (3). The CSCs which reattach throughout the body can then initiate a new tumor, resulting in metastases (4). Using current treatment methods capable of inducing cell death in the bulk of tumor cells, the CSCs are not destroyed due to their enhanced survival traits, such as quiescence and the expression of ALDH enzymes and ABC transporters (5). The remaining CSCs may then go on to recreate the original tumor, sometimes increasing the percentage of CSCs within the tumor and forming multiple drug resistant tumors (6). In other cases, the remaining CSCs will enter a state of dormancy within the body and remain undetected for long periods of time before reactivating and initiating the formation of a new tumor, thus resulting in cancer relapse in patients thought to be cancer free (7). As a result of these issues, new treatments are being investigated which can target CSCs. Natural products have shown the potential to induce cell death in CSCs, cause CSCs to differentiate, or sensitize CSCs to conventional chemotherapy treatments (8). Once the CSCs have been eliminated, the remaining tumor may diminish in size and can be subsequently eradicated through the use of conventional antineoplastic therapies (9).
According to the CSC model, cancer recurrence after treatment is due to the superior resistance of CSCs to cellular toxins and insults. While current treatments are capable of eradicating the bulk of the tumor mass, the lingering CSCs are able to form new, fully developed tumors from a small number of cells or even a single cell. CSCs are thought to resist treatment through several cellular mechanisms including the overexpression of drug efflux pumps, quiescence, and detoxifying enzymes [12
]. A high population of CSCs within a tumor has subsequently been linked to MDR and a poorer prognosis for cancer patients [13
]. Furthermore, the cellular machinery of CSCs has been shown to allow for epithelial-mesenchymal transition (EMT), a process by which epithelial cells lose their cell-to-cell and/or cell-to-matrix adhesion and can survive in a migratory state [14
]. By undergoing EMT, migrating to other organs, and reattaching by mesenchymal-epithelial transition (MET), CSCs are hypothesized to initiate the formation of metastatic tumors.
Current methods for the treatment of cancer have been demonstrated to be insufficient in eliminating CSC populations from a number of cancer types. Glioma CSCs have been shown to resist radiation therapy . Breast CSCs exhibit a similar resistance to radiotherapy in addition to common chemotherapy treatments [16,17]. Furthermore, the CSC population in residual breast cancer tumors has been shown to increase significantly following chemotherapy treatments, nearly doubling the tumorigenic potential of the residual cancer cells in immunodeficient SCID mice .
Epithelial-mesenchymal transition (EMT) is the process undergone by epithelial cells in which the cells alter their morphology, lose their polarity, and break cell-cell or cell-matrix adhesions. In this way, the cells gain mobility and invasive potential. EMT is an essential process during development and wound healing, allowing epithelial cells to produce a population of mobile cells able to migrate to target locations and reestablish basal and apical polarity once there . CSCs are hypothesized to possess enhanced EMT capability, enabling the cells to survive in the absence of cellular adhesion in addition to enhancing their resistance to apoptosis. CSCs having undergone EMT are thought to then reattach and produce metastatic tumors or circulate throughout the body in a dormant state, only to become active years later and cause distant cancer relapse to occur. The ability of CSCs to undergo EMT can be investigated by determining the expression of EMT related proteins such as Twist, Snail, or N-cadherin .
More commonly, however, EMT capability is assessed by removing any opportunity for cellular attachment. This can be accomplished through the use of non-adherent well plates, stirred bioreactors, serum-free growth conditions, or encapsulation in hydrogels. When in these conditions, cells without EMT capability will die leaving only cells that have undergone the transition. The remaining cells often grow in what are referred to as tumorspheres which have been shown to be enriched in CSCs in numerous tissues [49–51]. A major drawback of using these selective growth environments is the relatively low purity of CSCs in the resulting population. Further, CSCs within tumorspheres of a large enough size are likely to differentiate into phenotypically diverse cells. Still, drug discovery efforts directed at limiting the EMT capability of CSCs should be encouraged as this ability lies at the heart of the spread and recurrence of cancer that plagues many patients.
Limitation of Conventional Oncology
Treatments targeting a specific molecule or surface marker are likely to fail to eliminate CSCs due to the multiple survival pathways activated in CSCs in addition to the ambiguity of CSC markers across different tissue types, the presence of commonly used CSC markers in healthy tissues, and the often required combination of markers used to denote CSC populations. Treatments capable of reducing CSC populations will therefore require the development of novel, diverse, and multi-targeted approaches for cancer treatment. Conventional oncology however has of yet not found any safe and efficient targeted treatment. A chicken antibiotic called salymicin is pending in clinical trial and seems to be the most promising substance to target cancer stem cells.
Drug resistance in cancer stem cells
While resistance to chemotherapy treatments is not necessary to define CSCs, drug resistance is commonly associated with CSC populations. In fact, when resistance to a drug is induced, an increase in the percentage of cells possessing CSC markers has been observed . Resistance to specific chemotherapy agents in cancer cell lines is typically promoted in vitro by exposing the cells to gradually increasing doses of the drug or by exposing the cells to several cycles of clinically relevant chemotherapy doses followed by drug free media to mimic the treatment patients actually receive. The enrichment of CSCs following chemotherapy regimens observed both in vitro and in clinical studies  has enormous implications on drug discovery efforts and future cancer treatment. Without the ability to target and kill CSCs, chemotherapy treatments will continue to leave patients at risk for tumor recurrence and developed drug resistance. The following proteins and properties of CSC are thought to contribute to drug resistance in CSCs and therefore represent ideal targets for future chemotherapy or chemotherapy sensitizing drug discovery efforts. It is important to note, however, that healthy stem cells share many of the properties imparting drug resistance to CSCs, and as a result targeting these properties may lead to unwanted side-effects on otherwise healthy tissues.
ATP-binding cassette (ABC) transporters are transmembrane proteins that serve a crucial cytoprotective role for healthy stem cells throughout the body. The function of these proteins is to pump toxic compounds from the cell body before their deleterious effects can occur. These pumps are able to act on a large variety of compounds including many chemotherapeutic agents. The expression of ABC transporters has been used to indicate CSC phenotypes in multiple tissues and also plays a role in developing the multiple drug resistance (MDR) typical of CSCs . Members of the ABC transporter family that appear to be highly expressed in CSCs include, but are not limited to, ABCB1, ABCG2, and ABCB5 . The expressions of these proteins have been suggested as markers for CSCs, but the lack of appropriate antibodies makes their detection more difficult than previously discussed markers. The ability of the SP to exclude Hoescht 33342 is a result of ABC transporters, specifically ABCG2, making SP isolation an indirect method of CSC isolation based upon ABC transporter expression .
Many of the ABC transporter proteins have been “discovered” multiple times in the context of chemotherapy resistance leading to confusion in their identification. For example, ABCG2 is often referred to as breast cancer resistance protein (BCRP) alluding to its ability to confer MDR to breast tumor cells. ABCG2 expression has been identified in the drug resistant subpopulations of many cancer models including K562 chronic myeloid leukemia cells  and MCF7 breast adenocarcinoma to name a few . The cell lines in these experiments were made resistant through selection with various chemotherapies such as doxorubicin.
ABCB1 is another ABC transporter with multiple aliases. ABCB1 has been referred to by the names multidrug resistance protein 1 (MDR1), cluster of differentiation 243 (CD243), and most commonly P-glycoprotein 1 (P-gp). ABCB1 contributes to the efflux of many widely used chemotherapeutic agents including anthracyclines, vinca alkaloids, and taxanes making it a highly clinically relevant MDR protein . Reduction of the expression of ABCB1 has been shown to lead to an increased chemotherapy sensitivity of colorectal CSCs in addition to MDR cell lines of differing origin . By targeting ABC transporters, the unique resistance of CSCs can theoretically be reversed, sensitizing them to traditional chemotherapy treatments.
Another strategy CSCs employ in order to exhibit MDR is the rapid metabolization of the chemotherapy agents they are subjected to. As mentioned previously, the presence of ALDH enzymes and their activity is a commonly used marker to identify CSCs. ALDH enzymes exert their effect by oxidizing aldehyde groups of drug molecules, preparing them for future cell metabolism and thus detoxifying the cell. ALDH enzymes may also play a role in the differentiation of healthy and malignant stem cells. Inhibition of ALDH activity in ALDHhi/CD44+ putative breast CSCs convincingly resulted in a loss of MDR . Interestingly, the inhibition of ALDH activity using diethylaminobenzaldehyde (DEAB) further sensitized these CSCs to radiation therapy. By eliminating ALDH activity from tumors, the breakdown of chemotherapeutic agents within the tumor will be slowed resulting in a more effective treatment. Cytotoxic compounds which do not act as substrates for ALDH enzymes or that reduce their activity may have a unique ability to induce apoptosis in CSCs and act as more effective long-term treatments.
Pro-survival signaling and stem cell maintenance
CSCs hijack many of the pro-survival signaling cascades and maintenance proteins seen in healthy stem cells. In this way, CSCs have a tendency to survive cellular stresses capable of eliminating differentiated cancer cells in a similar fashion to non-malignant stem cells. For example, mechanistic target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) play a role in the maintenance and proliferation of healthy and cancer stem cells. The activation of phosphatase and tensin homolog (PTEN) and subsequent inhibition of mTOR and STAT3 results in a significant decrease in CSC viability and overall tumor drug resistance .
The stem cell maintenance proteins Wnt, Hedgehog, and Notch are also upregulated in CSCs. These molecules play a major role in maintaining the stem-ness of CSCs and activating the expression of stem cell related transcription factors such as octamer-binding transcription factor (Oct4) and Nanog as well as influencing EMT . Stem cell maintenance proteins such as these ensure CSCs will continue to asymmetrically divide, allowing the CSC phenotype to persist in a number of harsh conditions. Dysregulation of these pathways is hypothesized to promote gradual CSC differentiation leading to decreased tumor viability in response to chemotherapeutics, making them an attractive target for the treatment of both bulk tumors and CSCs.
Cellular quiescence is defined by a reduced occurrence of mitotic divisions within a cell population. Quiescence is recognized as a trait of most somatic stem cells, allowing them to survive in a state of relative dormancy and reduce the accumulation of DNA mutations over time . While debate remains as to whether or not chemotherapy agents have a diminished effect on quiescent cells, experiments on leukemia stem cells have shown that forcing these cells out of their dormant state results in increased drug sensitivity . The hypothesis behind this pathway for MDR is that diminished cellular metabolism, failure to proceed throughout the entirety of the cell cycle, and lack of DNA multiplication allows CSCs to avoid activating the targets of many chemotherapeutic toxins. Quiescence of CSCs not only potentially influences MDR, but also enables CSCs to remain dormant at the site of the original lesion or migrate throughout the body for years before attaching and initiating new tumors. Targeting the quiescence of CSCs has the potential to increase the efficacy of current therapeutic methods against CSCs within the original tumor as well as prevent CSCs from entering dormant states capable of initiating new tumors in patients in remission.
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