22 Apr 2020

Challenges and Promises of Targeting Cancer Stem Cells – the Proposed Achilles’ Heel of Cancer

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Introduction

According to the Canadian Cancer Society, nearly 1 in 2 Canadians will be diagnosed with cancer. Despite advances in diagnosis and treatment, cancer continues to be a death sentence for many patients due to relapse, metastasis at distant sites, drug resistance and the toxicities associated with select therapeutic treatment approaches [1, 2]. Studies in the past two decades have identified a subpopulation of cancer cells called cancer stem cells (CSCs) or tumour-initiating cells (TICs) as being solely responsible for tumour initiation, progression, relapse, metastasis and drug resistance [3-6]. Consequently, selective targeting of CSCs within the tumour cell population was initially thought to be a very promising therapeutic strategy to treating cancer [7, 8]. This essay will, therefore, elucidate on the characteristics of CSCs, the challenges and potentials of the proposed CSC therapy, and offer suggestions on the strategies to wholly targeting cancer to prevent relapse, metastasis and drug resistance.

Cancer Stem cells

Two models currently exist to account for tumour growth and the heterogeneity within tumours. The clonal evolution model argues that all cells within a tumour have the capacity to propagate all the various cell types in a tumour mass, and subclonal differences resulting from both genetic and epigenetic changes acquired during development are responsible for the intercellular variations [9]. Conversely, the CSC model suggests that all of the inherent characteristics of tumour, including initiation, heterogeneity, recurrence and metastasis, are being driven by a subpopulation of cells termed cancer stem cells [10, 11]. See Figure 1 below for an illustration of the two models. Although the first evidence of stem-cell like cancer cells was reported as early as 1937 [12], it was not until 1997 that Bonnet and Dick [13] provided indisputable evidence of the existence of a subpopulation of CD34+ CD38– cells in acute myeloid leukemia (AML) patients  that possessed the ability to self-renew, proliferate and differentiate into other cancer cells. CSCs were subsequently identified in a broad spectrum of human solid tumours, including breast (CD44+CD24–/low cells) [14], brain (CD133+ cells) [15], prostate (CD44+CD24– cells) [16, 17], among others. As one could imagine, identification of these CSCs was made possible due to specific cell surface markers they were expressing. It is, however, paramount to state that certain markers are not restricted to a particular tumour, and that cancers not expressing the associated markers above have been found to be also tumorigenic. To put this into perspective, CD133– cells in brain tumours have been found to possess high tumorigenic activity [18]. Apart from identifying CSCs using their cell surface markers by fluorescence activated cell sorting (FACS), other methods, including spheroid formation assay [19], side population assay [20] as well as a method based on the enzymatic activity of aldehyde dehydrogenase (ALDH) [21] have been used for the isolation and subsequent evaluation of CSCs.

Cancer stem cell model
Figure 1: A representative scheme of the two models that account for tumour growth and heterogeneity within a given tumour: the cancer stem cell model and the clonal evolution model. Figure adapted from [44].
There are considerable evidence that these CSCs have different cells of origin [22, 23], and that, depending on the cancer types, CSCs may originate from somatic stem cells, partially differentiated progenitor cells or differentiated cells that have acquired stemness through a number of dysregulated mechanisms [24, 25]. Certain signalling pathways have been attributed to the stemness phenotypes of CSCs, including, but not limited to, Wnt, JAK/STAT, Hedgehog, Notch and FAK signalling pathways [26, 27]. Figure 2 depicts a simple schematic representation of these different pathways and some of the targeting strategies that have been proposed to block these signalling pathways. The identification of these stemness propagating pathways, the characterisation of the properties of the CSCs themselves (promoting relapse and drug resistance, for example) as well as the isolation of specific cell surface markers as highlighted above have fuelled interests in designing targeted therapies solely against CSCs. The next section will focus mainly on the limitations and some successes that have characterised this ambitious goal.

Figure 2: Targeted therapies against dysregulated signalling pathways involved in CSCs: This schematic gives a broader knowledge of the different pathways that contribute to the stemness phenotypes of CSCc, including Wnt, JAK/STAT, Hedgehog, Notch and FAK signalling pathways. Figure also shows the subcellular localisation of the each of the signalling pathways, and specifically highlights the targeted therapies that have been designed to combat CSCs by directly inhibiting proteins or enzymes that function to promote the activities of these different signalling pathways. For example, WNT ligands and receptors can be inhibited by ipafricept and vantictumab, respectively. Although these agents are designed to inhibit CSC self-renewal, drug resistance and metastasis mechanisms, they are yet to be clinically demonstrated as being efficacious [27]. Figure adapted from [27].

CSC targeted therapies – a reality or an illusion?

Compelling evidence exists to suggest that targeted therapy has been somewhat successful in the clinic in the eradication of tumours. For example, olaparib, a PARP inhibitor, is used clinically to treat patients with metastatic HER2-negative breast cancers that also have mutations in the BRCA1 or BRCA2 gene [28]. But, as was previously proposed, would designing drugs targeting certain features that drive CSC phenotypes hold the cure to cancer? To answer this question, this section of the essay will discuss some of the limitations of targeting CSCs. First, although it may be possible to design drugs that target certain features of CSCs, it has been suggested that multiple CSC subsets, consisting of undifferentiated cells with different origins, may exist within a tumour. For example, studies suggest the existence of both CD133+ and CD133– CSC subpopulations with different origins [18, 29]. While targeted therapy against CD133+ CSCs may eliminate them, the CD133– subpopulation will be resistant [7]; thus, allowing the CD133– subpopulation to grow, divide and replenish the CSC population. Consequently, some monoclonal antibodies, including Lintuzumab against CD33, have been discontinued, despite showing some modest benefit [7, 30]. Another limitation to targeting CSC is that they share the same cell surface markers (e.g., CD133+) as normal stem cells [31]. Therefore, designing drugs against these markers will not be specific in targeting the CSC population within the tumour. For example, the selective CSC inhibitor, salinomycin, shows increased toxicity to normal CD4+ T cells at concentrations effective against CD4+ T cell leukemia cells [32].

Additionally, CSCs within certain tumours are known to possess a lower proliferative rate, display an elevated level of quiescence [33], and have efficient DNA repair mechanisms [34] compared to other cells within the same tumour. Consequently, these heterogeneous features of CSCs suggest that targeting them with drugs that damage DNA will not have a profound cytostatic effect on them. Compounding this further is the fact that CSCs also possess increased expression of anti-apoptotic proteins [35], ABC transporters (involved in increased efflux of chemotherapeutic agents) [36] and increased level of  ALDH and oxidant scavengers (both function to metabolise chemotherapeutic agents and reactive oxygen species [4]) that ultimately contribute to their resistance to chemo- and radiotherapy. Another hurdle in the CSC therapy is that eliminating CSC may not change disease outcome. This is because new CSCs are likely to be generated via the spontaneous dedifferentiation of non-CSCs due to cellular plasticity [37]. It would, therefore, be beneficial targeting both the differentiated cancer cells and CSCs with combinatorial therapies. Another factor that has limited the use of CSC targeted therapy is similarity in the many genes and signalling pathways that regulate both the stemness pathway and normal stem cells [27]. For example, proper regulation and homeostasis of intestinal stem cells are mediated via Wnt signalling [38, 39]. Hence, there have been concerns about toxicity and off-target effects of targeted therapies against dysregulated proteins in the signalling pathways. Finally, the efficacy of these targeted CSC therapies is not guaranteed due to redundancy in the signalling pathways [27]. To overcome this challenge, recent clinical trials are using high-throughput screening techniques to specifically target CSCs. The afore-mentioned challenges and limitations, therefore, weaken the claim that cancer can be cured by solely targeting CSCs.

Despite these hurdles or challenges, the subtle surface marker differences and the dysregulation of the signalling pathways in CSCs have been exploited as potential therapeutic targets [40]. Currently, some of these targeted therapies against CSCs have been investigated in different phases of randomised clinical trials (see table 1 below). For example, the Sonic Hedgehog pathway inhibitor, vismodegib, is currently being used to treat basal cell carcinoma [41] and has been evaluated to treat patients with Hedgehog pathway mutations that lead to medulloblastoma [42]. Majority of these studies are, however, combining with standard-of-care chemotherapy, and none have demonstrated clinical efficacy as single agent inhibitor [7, 27]. The preceding point illustrates the potential of using combination therapy to target both the CSCs and the differentiated tumour [43], thereby overcoming cancer cell heterogeneity and plasticity. Other therapeutic strategies that are currently being exploited in combating CSCs are depicted in Figure 3 below. While efforts have been made to specifically target CSCs with the aim to completely eradicate the tumour mass, as shown by the many targeted monotherapies, there have been no clinically approved single agent therapies against CSCs [27], suggesting that targeting CSCs might not improve clinical outcomes.

Figure 3: Targeted therapies against CSCs. Over the years, more targeted therapies against CSCs have been developed. These therapies have been classified into those that target signalling pathways that promote the CSC phenotypes (green area), targeted therapies against specific surface markers expressed by the CSCs (red area), drugs targeting ABC transporters that are known to be upregulated in CSCs (purple area) and lastly, drugs that inhibit certain growth factors and chemokines that collectively act to promote the CSC phenotypes. Figure adapted from [40].
BSC=best supportive care; carbo=carboplatin; CRC=colorectal cancer; AE= most severe adverse effect; etop=etoposide; GEJ=gastroesophageal junction; gem=gemcitabine; nab-p=nab-paclitaxel; NSCLC=non-small cell lung cancer; SCLC=small cell lung cancer. Table adapted (and modified to reflect current trial status) from [27].

Conclusions and future perspectives

Understanding tumour initiation, progression, and how to combat it has been the focus of cancer research for many decades. While the identification of CSCs and the elucidation of their signalling pathways have paved the way to targeting them, there are considerable challenges yet to be overcome as highlighted above. The idea of solely targeting CSCs is unrealistic given that they (CSCs) share so many characteristics with normal stem cells, together with the high intratumoural heterogeneity within CSCs. While studies evaluating monotherapies against CSCs have shown promise in early phase studies, combining anti-CSC therapies with other traditional or targeted therapies against not just the CSC subpopulation, but the bulk of the tumour represents one approach to eradicating cancers. In addition, there is the need to develop efficacious techniques to selectively target CSCs, while sparing normal stem cells. Finally, overcoming the challenge of CSC therapy resistance using innovative techniques, such as immunotherapy, represents another avenue yet to be investigated.

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