Understanding Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma: A Critical Update in Development of New Approaches

Cancer Stem Cells (CSCs) have an important role in Head and Neck Squamous Cell Carcinoma (HNSCC). HNSCC is considered as one of the major health problems throughout the world. These self-renewing cells are responsible for resistance to conventional anti-cancer therapy. CSCs have the ability of cancer recurrence; metastasis and can form a heterogeneous tumour. The idea of ‘CSCs’ has led the scientific community to a new era in the field of research and possibly effective treatment modalities for cancer in the future. This paper aims primarily to review the recent advancements made in the use of stem cells in the treatment of cancer. Secondly, this review presents a discussion on the consideration of CSCs being the backbone in the development of cancer and, precisely the role played by the CSCs in carcinogenesis and its outcome leading to development of possible cancer treatment in the future.

Medical databases including Pubmed Central, Google Scholar, Scopus, Copernicus, Science Direct, etc., were used to find all relevant articles related to HNSCC and its relation with CSCs, various mechanisms and therapeutic approaches. Various therapeutic approaches have been employed for the management of HNSCC such as surgical method, chemotherapies and radiotherapies and combinations or formulations of different drugs, but in most cases complete cure has been a failure. This is mostly because the CSCs escape such therapies. However, recent developments have led to the use of targeted therapy such as targeting cell surface markers or signalling pathways, targeting micro environment or blocking epithelial mesenchymal transition, immunotherapy and other approaches as well, leading to the complete eradication of CSCs in HNSCC successfully. Thus, this review presents a better understanding of CSCs and its mechanism of action for development of new treatment modalities.

Head and neck squamous cell carcinoma can be considered as solid tumours with heterogeneous content and usually have its origin in the epithelial tissue of the oral cavity, pharynx and larynx. A peculiar type of cancer cells has been found to be present in HNSCC and other tumours, similar in behaviour to tumour progenitor cells but having characteristic features consistent with CSCs [1,2]. The CSCs are defined as the cells in the tumour growth having a tumour initiating potential. There are three important characteristics of a normal stem cell:

As compared to normal stem cells, the CSCs are known to have no control on the cell numbers. A very small number of CSCs are responsible for the growth of the tumour cells. The identification of the cell type capable of sustaining the neoplastic growth is one of the fundamental problems in cancer. It is evident that most of the cancers are clones and each cancer cell represents the progeny of one cell, but the cell type (CSCs) possessing the tumour-initiating cell function and the methods to recognise them is still unclear [4].

Recently, the CSCs were also shown to be present in solid tumours such as breast cancer and brain tumours [5,6]. The CSCs not only have self-renewal capability but also can generate a wide spectrum of progeny like normal stem cells [7]. Therefore, newer therapeutic approaches are likely to be developed through the identification of CSCs in HNSCC that regulate the growth, metastasis, and treatment resistance of tumour.

Two important factors are considered to recognise the origin of the CSCs [Table/Fig-1]:

[Table/Fig-1]:

Schematic diagram depicting the origin of cancer stem cell (CSC). Normal stem cells produce normal progenitor cells which undergoes genetic mutation to be transformed into cancer cells.

Thus, CSCs can be obtained from either the self-renewing normal stem cells or from the progenitor cells with the ability of self-renewal due to mutations [10].

There are three possibilities of cancer stem cells playing a role in tumourigenesis [Table/Fig-2]:

[Table/Fig-2]:

Pictorial representation showing the outcomes of cancer treatment. Primary tumour cells undergoing chemotherapy can eventually lead to the development of chemo-resistant cancer stem cells leading to recurrence of metastasis.

The CSCs are comparatively quiescent than other cancer cells. The absence of activated hyperproliferation signals such as tyrosine kinase makes the CSCs resistant to the toxic anticancer drugs that usually target the rapidly dividing cells.

CSCs therapy includes cancer therapy by targeting the population of CSCs utilising several methods [Table/Fig-3]. Chemotherapy and radiotherapy are conventional therapy that are toxic to the healthy tissues and are not always able to kill CSCs, leading to multiple malignancies [12]. Moreover, by the process of Epithelial Mesenchymal Transition (EMT) in embryogenesis, CSCs can also promote metastatic characteristics of tumours [13-15]. Hence, CSCs therapy has become an important focus of cancer research to eliminate malignancies completely. There are various approaches that can target CSCs, most importantly the underlying cause that maintains it and can be utilised for new therapeutic strategies for head and neck cancer treatment. These approaches can be summarised as follows:

[Table/Fig-3]:

Diagrammatic representation of the current cancer therapies for head and neck squamous cell carcinoma targeting the population of cancer stem cells (CSC).

1. Targeting signalling pathways: The normal stem cells share common signalling pathways with CSCs like Hedgehog (Hh), Notch, Wnt/b-catenin, or High Mobility Group AT-hook 2 (HMGA2), B-cell lymphoma 2 (Bcl 2), B lymphoma Mo-MLV insertion region 1 homolog (BMI-1), and others that undergo aberrant activation or dysregulation to give rise to CSCs [16-18]. Amongst these, BMI-1 is considered as one of the important stem cell-related genes involved in the regulation of carcinogenesis in head and neck cancers. It was reported that the chemosensitivity of CSCs in HNSCC improved after knockdown of BMI-1 and CD44. Furthermore, the sensitivity of HNSCC cells increased to cisplatin on knockdown of CD44; thus, accounting for CSCs in response to chemotherapy [19]. This suggests that those anticancer drugs having the formulations of various inhibitors of such signalling pathways can be an answer to target the CSCs. However, the drawback of using such inhibitors is their adverse effect on the normal stem cells. Therefore, a combination of other CSCs-targeting therapies can also be used in anticancer drug formulations in order to improve their specificity.

2. Targeting detoxifying enzymes and drug efflux pumps: Certain CSCs are rich in drug detoxifying enzymes like Aldehyde Dehydrogenases (ALDH)-1. The importance of ALDH1 is its specificity as CSCs marker for the identification of highly tumourigenic cells present in HNSCC [20]. It has been shown that ALDH1+ CD44+ cells in HNSCC cells promote tumour propagation by resistance to radiotherapy and maintaining CSCs like properties [21]. Studies have also shown that the use of specific ALDH inhibitor Diethylamino-Benzaldehyde (DEAB) or All-Trans Retinoic Acid (ATRA) caused inhibition of ALDH1 activity in breast CSCs, resulting in reduced aggressiveness. Thus, the sensitivity of breast CSCs was increased towards chemotherapeutic drugs [22]. It was demonstrated earlier that the survival fraction of CSCs was decreased by UCN-01 (a checkpoint kinase inhibitor) in combination with ATRA with irradiation, thus suggesting for a powerful radio-sensitising strategy in HNSCC [23]. Moreover, the ATP Binding Cassette (ABC) drug transporter proteins common for both normal and CSCs are efflux pumps and protect the cells from xenotoxins. These pumps offer multiple drug resistance to the CSCs. It also has been reported that in lung cancer cells the use of pharmacological molecules in vitro and in vivo to target the ABC drug transporter proteins has led to increased sensitivity of CSCs to chemotherapy and radiotherapy [24].

3. Targeting CSCs molecules using micro-RNA: CSC markers can directly target certain micro-RNAs for their down-regulation. For example, miR-34a is one such micro-RNA which is a key negative regulator of CD44+ prostate cancer cells. This suggests the use of miR-34a as novel therapeutic agent against prostate CSCs [25].

4. Induction of CSCs apoptosis and differentiation: The resistance to chemotherapy and radiotherapy can be produced by anti-apoptotic proteins in CSCs such as Interleukin-4 (IL-4) produced by CD133+ colon carcinoma cells [26]. Experiments have shown that the sensitivity of CD133+ colon carcinoma cells to chemotherapeutic drugs (oxaliplatin and 5-fluorouracil) increased successfully by the use of IL-4 neutralising antibody and IL4 antagonists to target IL-4. Inducing the terminal differentiation of CSCs by agents like ATRA and retinoids, Histone Deacetylase Inhibitors (HDAI) and Bone Morphogenetic Protein 4 (BMP 4) can also be an approach to inhibit the self-renewal of CSCs instead of killing CSCs [27-29].

The origin of tumour may be accompanied by a deorganisation of the normal cellular structure of the tissue/organ and/or an increased proliferative potential known as anaplasia [30]. A number of morphological characteristics are usually considered to define anaplasia, including the following:

1. Resistance to chemotherapy: There are various factors that contribute to drug resistance in cancers for e.g., glutathione and its enzyme structure like topoisomerase II, O6-methylguanine-DNA-methyltransferase, dihydrofolate reductase, metallothioneins, and ABC transporter proteins which are encoded by the multidrug resistant gene, the multidrug resistant protein, and the breast cancer resistant protein1 [31,32]. Therefore, it becomes crucial to investigate relationship between CSCs and these factors.

2. Resistance to irradiation: The ability of cancerous cells to survive and cause tumour recurrence suggests the property of radio-resistance of CSCs [33].

3. Invasion/metastatic activity: The ability of the malignant tumour cells to invade and disseminate into normal tissue help them to metastasise into other tissues. CSCs have high invasion activity as evident from the fact that even surgical operations cannot remove some of the infiltrating cancer cells and causes recurrence. This is also supported by the higher expression of CD44 and chemokines. Chemokine (C-X-C motif) receptor 4 (CXCR4) mediates cell migration in CD133+ cancer cells [34,35].

1. Cell surface markers: A useful way to separate CSCs from many types of tumours is by cell surface markers, such as CD133. Generally, CD133 molecule (a transmembrane pentaspan protein) is used as a marker of normal haematopoietic stem cells and organ-specific stem cells [36]. The expression of CSC markers in cancers differs in patterns, histological types and degrees of differentiation. The benefit of using such markers is the isolation of CSCs by Fluorescence-Activated Cell Sorting (FACS) and analysis of their biological characteristics for therapeutic purposes.

2. Sphere formation assay: The sphere forming methods are used by CSC researchers to concentrate CSCs in culture. However, it is better to use a monolayer culture method to characterise CSCs. The monolayer-cultured CSCs provide the advantage of being expanded as a homogeneous population [37].

3. Aldehyde dehydrogenase activity: ALDH is a detoxifying enzyme which blocks alkylating agents by oxidising intracellular aldehydes to carboxylic acids. It has been shown earlier that ALDH increases in Tumour Stem Cells (TSCs) [38,39]. Increasing evidence is available which reports that ALDH is expressed strongly by many types of CSCs and can be purified from tumours and cancer cell lines [40-42].

4. Side population: Studies have shown the presence of TSCs in both Side Population (SP) and non-SP and that stem cell marker are not expressed by SP cells [43,44]. It has been also demonstrated that only SP cells and not the non-SP cells possess the property of self-renewal in culture resistance to anti-cancer drugs including mitoxantrone, and formation of tumours when transplanted in vivo [45-50].

5. Niche for CSCs: The regulation of TSCs involves both intrinsic mechanism and extracellular signals which are derived from specialised microenvironment “niche”. This is proved by the fact that the ablation of such niche results in loss of TSCs. It appears that such niche is also required by CSCs for tumourigenesis. A premetastatic niche is formed by the bone-marrow derived progenitors before the arrival of cancer cells in the tumour specific premetastatic sites. This is evident from the fact that the ablation of such niche prevents tumour metastasis [51]. Experiments involving specific ablation of endothelial cells associated with tumour using inducible caspase-9 in Severe Combined Immunodeficiency (SCID) mouse model of human tumour angiogenesis showed that there is a decrease in fraction of head and neck CSCs [52]. Similarly, it has been also demonstrated that targeting the CSC niche produced more durable response in HNSCC; thus, developing a new concept of using both conventional chemotherapy and CSCs targeted therapy [53].

Genetic alterations cause maintenance of TSCs, amplification of precursors, or transformation of differentiated cells to CSCs.

1. p53 pathway: Studies have shown that loss of p53 function promotes accelerated cell proliferation and malignant transformation [54]. The p21 cyclin-dependent kinase (cdk) inhibitor which is considered as the effector of p53 regulates the progression of cells through the G1 cell-cycle phase. However, the use of p21 gene itself as an oncogenic target in human cancers has not been demonstrated.

2. Activation of receptor tyrosine kinase pathway: Signalling pathways of Receptor Tyrosine Kinases (RTKs) that play a role in maintenance of TSCs and amplification of precursors such as Platelet-Derived Growth Factor Receptor (PDGF), Epidermal Growth Factor receptor (EGF), fibroblast growth factor receptor, and insulin-like growth factor 1 receptor are frequently mutated in tumours [55].

3. Notch signalling pathway: Receptors play an important role in biological functions such as cell proliferation, differentiation, survival, and tumourigenesis [56]. Evidences are available which suggest that apart from maintaining the multi-potentiality of neural stem cells the notch activation plays a role in tumourigenesis.

4. Wnt signalling pathway: There are diverse developmental processes like cell proliferation and fate decisions that involves coordination of Wnt family of secreted proteins [57-59].

5. Hedgehog signalling pathway: The processes like proliferation, development, and tumourigenesis also involve Hh signalling. Studies have shown the involvement of ectopic activation of Hh signalling in tumour formation in the central nervous system (CNS), thus suggesting an important role of Hh signalling in brain tumourigenesis [60-62].

6. Stemness in tumour cells: The adult stem cells have a relatively long half-life and can suffer prolonged exposure to genotoxic stress during tumour development. This may cause accumulation of initial mutations leading to cancer [63].

7. Biomarkers in CSCs: There exists a difference in the expression of CSCs markers in cancers depending on its pattern, histological types and degrees of differentiation. Such markers are beneficial in the isolation of CSCs and analysis of their biological characteristics for effective therapeutic purposes.

8. CD133: Studies have shown the use of CD133 as a surface marker of CSCs in solid primary tumours like medullo-blastomas and glioblastomas, as well as cancers of epithelial tissues. CD133 molecule (a transmembrane pentaspan protein) is also considered as a universal marker of normal haematopoietic stem cells and organ-specific stem cells [64].

9. CD44: Several functions are attributed to the CD44 protein such as cell adhesion, motility, proliferation, drug resistance, and cell survival [65,66]. Experiments have also shown the role of CD44 in lymphocyte homing, wound healing, and cell migration, cancer cell growth and metastasis [67,68]. An immune-deficient mouse model was used in one of the first studies on CSCs in HNSCC. This study demonstrated that those CD44+ cancer cells that are responsible for formation of new tumours in vivo along with the ability of self-renewal and differentiation constitute only 10% of the cells in a HNSCC primary tumour. However, some of HNSCC expressing CD44s (standard form) and CD44 v6 (alternative splice variant), particularly in laryngeal cancers are associated with a poorer disease-free survival [69].

10. CD24: Recent studies have reported the role of CD24 in the developmental hedgehog pathway that is often active in CSCs [70].

11. CD138: Tumour cells that lack CD138 are capable of clonogenic growth and are relatively drug resistant. Such functional properties have also been attributed to CSCs in several human cancers [71].

12. Other CD molecules: Additionally, a list of other CD molecules in CSCs in various types of cancer is provided in the [Table/Fig-4] [72-79].

A better understanding of the resistance mechanisms of CSCs in HNSCC is required involving future studies for improving therapy and possibly preventing tumour spread or recurrence. The important roles of CSCs in progression of cancer can be evaluated by complementary in vitro approaches. Investigation of CSCs gives the idea of making novel targets for cancer that can be helpful in drug resistance and may combat the tumour cell metastasis [80]. When the patients are given therapy to cancer, great care must be taken as cancer stem cells are similar to normal stem cells. New and improved experimental approaches such as in vitro assay systems, will allow scientists to evaluate the relation between the different cell types within a tumour and their microenvironment.

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