Cancer stem cells (CSCs) have been implicated in the processes of metastasis, tumour recurrence, and drug resistance so it is not surprising that they have become a major focus of cancer research in recent years. However, it remains to be seen whether CSCs can be targeted successfully, and if so, without adversely affecting normal stem cells within the body. This review highlights the evidence for the existence of CSCs and their potential use as a chemotherapeutic target in the future. In discussing the therapeutic potential of targeting CSCs, it is necessary to examine the CSC hypothesis and the biology of CSCs, focusing on their ability to evade conventional chemotherapeutic agents. The evidence reinforces the theory that drugs may be required in the treatment of cancer which target CSCs, killing them before drug-resistant tumours may metastasise or tumour recurrence occurs. CSC-targeting agents should ideally be used in combination with conventional chemotherapeutics which target the rapidly proliferating, primary neoplastic cells. However, more research needs to be done on the complexity of CSC cellular processes before this type of drug can be used clinically.

Introduction

The treatment of cancer has come a long way in the last number of decades. Scientists have made massive leaps in both understanding tumour biology and developing drugs which target cancer cells. Chemotherapy has hugely benefited cancer patients by shrinking their tumours, reducing incidence of metastasis and relapse, and improving their overall survival. It is true, however, that conventional chemotherapy works better for some forms of cancer than others, and it is not without its limitations or side effects. Numerous recent studies have suggested that cancer stem cells (CSCs) are responsible for many of the limitations of conventional chemotherapy, such as therapeutic refractoriness and disease relapse. As chemotherapy frequently targets actively proliferating cells, residual dormant cells are often left behind. These quiescent cells, or CSCs, are largely responsible for any metastasis, tumour recurrence, or drug resistance that may occur.1 CSCs can also overcome chemotherapy by accumulating mutations that lead to drug resistance. Therefore, drugs which target CSCs, in addition to primary neoplastic cells, are required so that these boundaries can be overcome.

What are cancer stem cells?

The stochastic evolutionary model suggests that tumour growth is a random process in which all cells within the tumour can contribute. However, the CSC hypothesis postulates that there is a subpopulation of cells within a tumour that are similar to normal stem cells and have the ability to divide asymmetrically.[1] In recent years, it has become clear that many tissues, both malignant and non-malignant, are arranged in a Stem-Progenitor-Differentiated cell hierarchy. In this arrangement, the dormant CSCs sit at the apex and differentiate into progenitor cells, which then produce differentiated neoplastic cells. The CSCs can then return to their quiescent state.[2] This hierarchy allows CSCs to have a heightened ability over differentiated neoplastic cells to generate new tumours, a process known as tumour seeding. The first evidence for the existence of CSCs came to light in the 1960s. Kleinsmith and Pierce demonstrated that a single embryonal carcinoma cellwas capable of seeding new tumours when transplanted into SCID mice.[3] However, the term “cancer stem cells” was not introduced until 2001. Since then, there has been increasing evidence to support the CSC hypothesis, with CSCs being discovered in many other forms of cancer.1 Although controversial, the self-renewal of CSCs may promote tumour growth, and understanding CSC biology may allow the identification of attractive therapeutic targets.

The origin of CSCs is not fully understood. Research has shown that CSCs involved in carcinoma arise, at least partially, due to a process known as epithelial-mesenchymal transition (EMT).[4] EMT is an important process in embryogenesis and organogenesis but occurs pathologically in the setting of carcinoma. Various signalling pathways (e.g., Wnt signalling) induce the EMT pathway in epithelial cells and cause expression of a series of transcription factors which repress epithelial genes and activate mesenchymal genes.[5] During this process, epithelial cells lose their characteristic phenotype and gain features of mesenchymal cells including increased motility, invasiveness, and resistance to apoptotic factors, permitting dissemination of carcinoma. Certain types of EMT have also been shown to allow some cells to exhibit traits of CSCs, such as self-renewal. Mani et al. showed that human mammary epithelial cells which had undergone EMT expressed surface proteins similar to those found on breast CSCs.[4] These cells also displayed a heightened ability to seed new tumours, verifying the generation of CSCs. It has also been theorised that CSCs may arise due to mutations in normal stem cells or progenitor cells.  Driessens et al. observed that progression from benign papilloma to malignant squamous cell carcinoma in mice is associated with an expansion of the CSC population and a decrease in the production of non-stem cells.[6] This study suggests that the most likely source of CSCs are normal stem cells that accumulate mutations.

CSCs are believed to be more resistant to conventional therapies in comparison to other cell populations located within the tumour.[1,7] This resistance has obvious important clinical implications regarding tumour relapse. Understanding the mechanisms underlying resistance is imperative if promising therapeutic targets are to be identified. It has been demonstrated experimentally that human mammary epithelial cells induced into an EMT have increased resistance to radiotherapy and chemotherapy.[8] Following EMT induction, these cells showed a 10-20 fold increase in resistance to common chemotherapeutic agents such as paclitaxel, doxorubicin, and dactinomycin. The mechanisms by which EMT enhances this resistance are unknown. The slow turnover of CSCs compared to other neoplastic cells, and their increased quiescence, are other obvious mechanisms of resistance to chemotherapeutics targeting rapidly-proliferating cells.[2] Additionally, CSCs express high levels of anti-apoptotic proteins such as Bcl-2, as well as ATP binding cassette (ABC) efflux pumps, which are both associated with drug resistance.[9] The cytoplasmic enzyme aldehyde dehydrogenase is thought to be involved in CSC resistance in colorectal tumours to cyclophosphamide.[7] This enzyme oxidises and inactivates the toxic product of the drug, aldophosphamide. Dylla et. al. discovered that treatment of colorectal cancer with cyclophosphamide results in survival and enrichment of CSCs, resulting in the rapid regeneration of tumours expressing increased levels of oncogenes.[7] This selective outgrowth of drug-resistant cells highlights the role of CSCs in the relapse of multi drug-resistant tumours.
 

How can we develop drugs which target CSCs?

Although research on the formation and resistance of CSCs has led to a greater understanding of how drugs targeting these cells could potentially be developed, experimentation on CSC-targeting drugs is very problematic, largely due to the difficulty in isolating CSCs and growing them in culture.[10] This makes it very difficult to test agents for specific toxicity against them. One method of overcoming this obstacle is to induce epithelial cells into an EMT. As already discussed, this enriches cells with CSC-like properties, including increased chemotherapeutic resistance.[4] Screening for agents with selective toxicity against these cells can then be performed. One experiment by Gupta et al. screened approximately 16,000 compounds for selective toxicity against EMT-induced CSCs. Only 32 of the 16,000 substances displayed any selective toxicity against these cells, of which only four were found to be suitable for further study. Salinomycin was established to be the drug with the greatest potential. Further investigations revealed that salinomycin inhibited 100 times more breast CSCs than paclitaxel, the major conventional chemotherapeutic used to treat breast cancer.[8] However, salinomycin is very toxic to the body and could potentially cause serious side effects in vivo.[11]

Another study by Visnyei et al. looked at drugs which target CSCs in glioblastoma.[12] This study took a different approach to trigger CSC enrichment by culturing glioblastoma samples in neurosphere media containing suitable growth factors. This medium triggered an enrichment of CSCs, while a control medium did not. Approximately 30,000 compounds were then screened to see if they adversely affected the proliferation and survival of glioblastoma CSCs, of which four selectively toxic compounds were used to pre-treat glioblastoma CSCs before implantation into SCID mice. The pre-treated CSCs produced a substantially decreased tumour mass compared to untreated CSCs. This reduction in tumour mass occurred due to specific loss of glioblastoma CSCs following treatment with the compounds. Although the cells were cultured in an in vitro environment, the tumours that formed were genetically and phenotypically identical to the original glioblastomas. However, it is not known if in vivo administration of these drugs would be safe or effective. Therefore, developing drugs which interfere with specific CSC cellular processes could prove to be more effective at causing their selective destruction in vivo.

Perhaps the technique with the most potential is blocking cellular processes involved with induction and maintenance of the stem cell state. For example, it has been discovered that epithelial cells secrete inhibitors which block pro-EMT factors such as Wnt proteins and TGF-beta, preventing entrance into EMT under normal physiological conditions.[5] Therefore, derivatives of these molecules could potentially reduce the proportion of CSCs in a tumour. However, it is very difficult to target Wnt signalling in CSCs without targeting normal stem cells involved in homeostasis. For example, in animal studies, the inhibition of Wnt signalling in colorectal cancer resulted in severe defects in intestinal development.[13] The Hedgehog signalling pathway has also been implicated in a number of CSC cellular processes, particularly cell self-renewal.[14] This has been characterised in a number of cancers including chronic myeloid leukaemia, pancreatic adenocarcinoma, and multiple myeloma.[15-17] Cyclopamine is an active compound capable of inhibiting the Hedgehog pathway, and is therefore a potential CSC-targeting drug.[18] A derivative of this drug has entered clinical trials and has exhibited anti-tumour activity against basal cell carcinoma.[19] A derivative is also being used in clinical trials in combination with gemcitabine for the treatment of pancreatic adenocarcinoma.[20] However, it remains to be seen whether this type of drug will affect CSCs without adversely affecting the normal stem cell population within the body.

A recent interesting study by Song et al. has identified mitochondria as potential drug targets in CSCs.[21] Cancer cells are more susceptible to mitochondrial injury due to their extensive metabolic rearrangements.[22] Mitochondria are thus a potential target for inducing apoptosis of multi-drug resistant CSCs. Hirsch et al. discovered that the anti-diabetic drug metformin leads to the selective destruction of breast CSCs. It is thought that inhibition of the mitochondrial protein Complex I (NADH Dehydrogenase) leads to inactivation of a stress response and resultant inactivation of the pro-inflammatory transcription factor NF-κB. Since CSCs have a heightened inflammatory regulatory circuit, breast CSCs are selectively killed.[23] This could explain why diabetic patients taking metformin have a reduced incidence of a multitude of cancers.[24] It was also discovered that a combination of metformin and doxorubicin accelerates CSC apoptosis in mouse xenografts compared to doxorubicin monotherapy.[25] Another study by Alvero et al. used an isoflavone derivative to target mitochondrial function in ovarian CSCs.[26] This led to a decrease in concentrations of ATP, Complex I, and Complex IV in CSC mitochondria, as well as an increase in the concentration of reactive oxygen species. Following this, activation of two distinct cellular pathways culminated in the induction of CSC apoptosis. Although targeting CSCs in this manner is very exciting, there is potential for serious side effects due to the sharing of metabolic pathways and cell surface markers between CSCs and normal stem cells within the body.[21] Therefore, a more in-depth understanding of mitochondrial function and CSC metabolism is required.

An alternative method of targeting CSCs is to induce their differentiation into primary neoplastic cells, making them susceptible to conventional chemotherapeutic agents. This must be done without inducing the differentiation of normal stem cells. Sachlos et al. investigated whether neoplastic haematopoietic stem cells (HSCs) in acute myeloid leukaemia (AML) could be differentiated without inducing the differentiation of normal HSCs within the bone marrow.[27] After screening a wide range of compounds, treatment with the anti-psychotic drug thioridazine was found to selectively induce this differentiation. This drug acts via a dopamine receptor and appears to work by reducing the levels of Oct4 in CSCs, a transcription factor that is involved in maintaining stem cells in an undifferentiated state and promoting self-renewal.[28] The drug also upregulates genes which are specifically involved in the differentiation of neoplastic HSCs.[27] Therefore, thioridazine induces differentiation of CSCs in AML but has little effect on normal HSCs. Since thioridazine acts via a dopamine receptor, it is likely that neoplastic HSCs express this receptor, while normal HSCs do not. Therefore, this may be a likely candidate which could be targeted by selective agents in the future.

Immunotherapy has become an exciting avenue in the treatment of many different forms of cancer in recent years. These new “magic bullet” type drugs have become the focus of huge interest in the scientific community and the public alike. Drugs like ipilimumab used for the treatment of melanoma have revolutionised the way in which we treat some cancers.[29] Therefore, it is not surprising that efforts are being made to use immunotherapy to target CSCs. This form of treatment could work by using one of the unique cell surface markers expressed by CSCs as a target for the patient’s own immune system. There are challenges to this type of therapy, however, particularly the autocrine secretion of anti-inflammatory proteins by CSCs, namely TGF-beta.[5] Nonetheless, the development of monoclonal antibodies that target the unique cell surface markers should be explored in the future. For example, CD133 is highly expressed in many different CSCs, including those of glioblastoma, lung cancer, and breast cancer.[30] Therefore, treatments targeting these CD133 markers may be an alternative therapeutic option. Additional research is required in order to identify specific cell surface markers and exploit them therapeutically.
 

Conclusion

Can CSCs be used as a chemotherapeutic target?

Although conventional chemotherapy has greatly improved the prognosis of cancer, it has many limitations which must be overcome with new therapeutic options. This fact is highlighted by newer cancer treatments such as immunotherapies, which have significantly improved the prognosis for patients with a wide variety of cancers. However, targeting CSCs may be a more efficacious alternative. In order to develop such drugs, further research regarding the metabolic pathways unique to CSCs and the influence of their tumour microenvironment is required. Despite the need for further research, many potential strategies for targeting CSCs have been identified in the last number of years. These include inducing CSC differentiation into normal neoplastic cells, interfering with unique metabolic pathways, and targeting unique cell surface markers. Of course, there is still some work to be done before drugs like this become available in the clinic, but recent advances have shown that this is an exciting therapeutic option that we will no doubt be hearing more about in the future.

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