Cancer Stem Cells in Head and Neck Squamous Cell Carcinoma

ABSTRACT

Cancer stem cells (CSCs) are a small sub-population of cells within a tumor mass proficient of tumor initiation and progression. Distinguishing features possessed by CSCs encompass self-renewal, regeneration and capacity to differentiate. These cells are attributed to the phenomenon of aggression, recurrence and metastasis in neoplasms. Due to their cancer initiating and contributing features, a proper understanding of these CSCs and its microenvironment would aid in better understanding of cancer and designing better targeted therapeutic strategies for improved clinical outcome, thus improving the prognosis. This article dispenses a narrative review of CSCs in the context of head and neck carcinoma under the sub headings of overview of cancer stem cells, methods of isolation of these cells, putative CSC markers of head and neck cancer, signaling pathways used by these cells and their therapeutic implications.

INTRODUCTION

Head and neck cancers encompass an array of neoplasms arising from the oral cavity, nasopharynx, oropharynx, larynx and hypopaharynx, each characterized by their distinctive etiology, epidemiological trends and treatment modalities.[1] Among the varied entities, head and neck squamous cell carcinoma is the sixth most common cancer occurring worldwide, while squamous cell carcinoma is the most common type of malignancy encountered in the oral cavity.[2,3] In spite of the advancements in the field of treatment, the survival rate of Head and Neck Squamous Cell Carcinoma (HNSCC) has not improved considerably over the years, and the prognosis remains poor.[4] Researches have suggested that the phenomenon of aggression, recurrence and metastasis in these neoplasms could be attributed to the cancer stem cells (CSCs).[5]

Two main hypothetical models have been put forward to explain the origin, amelioration and reoccurrence of neoplasms. According to the stochastic model, the cells constituting a tumor are homogeneous in nature, and each cell has the capacity to initiate tumorigenesis. All the cells have equal proclivity to begin, propagate and disseminate a neoplasm.[6] However, accretion of numerous discrete mutations and micro environmental signals provides fringe benefit to few tumor cells.[7] A randomly occurring genetic mutation which could affect any cell is responsible for malignant transformation. These cells proliferate, genomic instability accumulates, post which the most aggressive sub clones are selected resulting in tumor evolution. Spontaneous shifts in cell phenotype resulting in sub clone formation explains the heterogeneity of tumors according to this model.[8] On the other hand, cancer stem cell hypothesis puts forward a hierarchical model of initiation and progression of neoplasms.[6] CSCs are a miniscule sub population of cancer cells, which are self-sustaining, and possess the exclusionary ability to divide and result in the diversified descent of cancer cells that constitutes the neoplasm. These cells are capable of initiating cancer, and hence also referred to as “tumor initiating cells”.[9] Besides these cells, the majority of neoplasm is composed of post-mitotic differentiated cells and rapidly dividing cells, called transit amplifying cells, which are derived from differentiation of CSCs and lack the potentiality to commence tumorigenesis by themselves.[6]

Researches over the year have shown that both the theories are not inconsonant, rather are tumor-type-specific and have different roles to play in different stages of progression of tumor.[6] Both the models explain certain aspects of HNSCC. Stochastic model annotates the presence of wide ranging pre-neoplastic areas further from the surgical margins, which are responsible for recurrences and secondaries. Contrarily, cancer stem cell hypothesis explains the heterogeneity in HNSCC and distant metastasis.[8,9]

CONCEPT OF CANCER STEM CELLS

The CSC model contemplates tumor as an abnormal organ comprising of echelon of cells, inclusive of self-renewing stem cells and highly proliferative progenitor cells, which differentiate and form the majority of tumor mass.[10] The key features of this hypothesis include: of the entire tumor mass, very few cells are potent to initiate tumorigenesis when transplanted to an immunodeficient mice; specific surface markers can distinguish cancer stem cells from other cancer cells; tumors originating from the CSC population accommodates heterogeneous population of tumorigenic and non-tumorigenic cells of the original tumor; and, CSCs are a group of self-renewing cells, which can be transplanted through multiple generations.[5]

Distinguishing features possessed by CSCs include tumor initiation, self-renewal, regeneration and capacity to differentiate. Moreover, these cells also show notable resistance towards chemotherapy and radiotherapy, thus warranting the need to change the current treatment measures which focuses on comprehensive abatement of tumor mass.[11] In addition to these features, CSCs also help in metastatic spread of tumors by facilitating epithelial-mesenchymal transition (EMT).[12] These CSCs if not targeted during treatment, can result in the tumor to relapse or metastasize, and worsen the prognosis.

CSCs have been theorized to originate from normal stem cells or progenitor cells undergoing genetic alteration, dedifferentiating and acquiring the features of cancer stem cells. These may also stem from the amalgamation of a hematopoietic stem cell with a mutated somatic epithelial cell or from dedifferentiation of a mature cell. In addition, dedifferentiated somatic cells or cancer cells may undergo epithelial mesenchymal transition to form CSCs.[13] Stem cells rely on their surrounding immediate microenvironment or niche for viability and functioning, which is comprised of non-epithelial stromal cells, inflammatory cells and cells constituting the vasculature, all of which bolster and sustain the CSCs.[14] Moreover, the niche components inkle the proliferative and self-renewal signals, which aids in maintenance of the undifferentiated state of these cells. Studies have suggested the extant of a perivascular CSC niche in HNSCC and the dependence of their interactions with the niche components for their survival.[15]

HOW NORMAL STEM CELLS DIFFER FROM CSCS?

Normal stem cells (NSCs) have extensive, but limited self-renewal capacity, while CSCs have indefinite self-renewal capacity.[16] In addition, NSCs and CSCs differ in their degree of dependence on the stem cell niche, the specialized microenvironment wherein the stem cells reside. CSCs may involve deregulation or alteration of the niche by dominant proliferation- promoting signals whereas NSCs support and are supported by the niche which maintains a homeostasis.[17] NSCs contain a normal diploid genome and are stable, but CSCs are aneuploid with chromosomal rearrangements. Moreover, NSCs are generally quiescent or very slow growing, reside in a specific niche, and have relatively long telomeres. Contrarily, CSCs expressing stem cell markers are not completely quiescent, and have short telomeres which is a hallmark of cancer.[18] CSCs have been reported to be resistant to conventional radiation and chemotherapy treatment, but NSCs are not. NSCs have the capacity to generate normal progeny with limited proliferative potential, while CSCs can produce phenotypically diverse progeny.[16]

CSC BIOMARKERS IN HNSCC

27 molecules enciphered by 28 genes have been delineated to be closely associated with the cancer stem cell properties in HNSCC, of which four are found in the cell membrane (CD44, EpCAM, CD10, TAZ), four in the cytoplasm (CD24, MT1-MMP, ALDH1, GRP78), 12 in the nucleus (topoisomerase I/IIα/IIIα, Notch1, Brachyury, ABCG5, Sox2, SLC2A13, Nanog, KLF4, JMJD6, EHMT2), three in both the cytoplasm and cell membrane (c-Met, CD133, CD166), two in cytoplasm and nucleus (Oct4, Bmi-1) while one in both cell membrane and nucleus (ABCG 2). In addition, CD38 is distributed at all the three sites i.e cytoplasm, nucleus and cell membrane.[19]

These molecules have been broadly categorized into six groups based upon their functional roles: regulation of replication (topoisomerase I, IIα, IIIα), regulation of transcription (TAZ, Oct4, Bmi-1, KLF4, Sox2, Nanog, EHMT2, Brachyury), signal transduction (CD24, EpCAM, c-Met, CD133, CD44, CD166, CD98, Notch1), transportation (SLC2A13, ABCG2, ABCG5), enzymatic action (MT1-MMP, ALDH1, CD10, JMJD6) and chaperone protein (GRP78).[19]

Cluster differentiation 44 (CD44) - A surface glycoprotein, functioning as hyaluronic acid receptor, actively participates in cell migration and adhesion. It is amongst the credible markers for HNSCC CSCs.[20] Studies have shown that CD44 is involved in tumor initiation, progression, metastasis and plays an indispensable role in growth, survival, differentiation and migration.[21] It has also been found to play significant role in apoptosis inhibition, collagen degradation, invasion and neovascularization, by binding to growth factors and matrix metalloproteinase MMP9, thus facilitating cancer spread.[7] Overexpression of CD44 is kindred with regional lymph node metastasis, perineural invasion, increased loco-regional recurrence, increased radiotherapy resistance, decreased overall survival and thus, poor prognosis.[22] The use of CD44 alone as a marker is contentious as it is explicitly seen in normal head and neck epithelium. Nonetheless, when used in combination with other markers, it could be useful in identification of CSC population.[23]

CD133 - This transmembrane glycoprotein, also known as Prominin-1 has been associated with amplified clonogenicity, an EMT phenotype, tumor sphere formation, self-renewal, proliferation, multilinear differentiation, invasion and tumorigenicity in HNSCC cell lines. This marker is positively correlated with poor overall survival and prognosis in HNSCC patients.[6,24] It has also been hypothesized that CD133 is crucial to modulation of chemosensitivity, and can be used as therapeutic targets in drug development.[25]

ALDH - This intracellular enzyme is expressed in many progenitor and stem cells, with its primary function being conversion of retinol into retinoic acid during early stem cell differentiation and catalysis of oxidation reaction which converts toxic intracellular aldehyde metabolites into carboxylic acid. ALDH is confederated with spheroid formation, tumor formation, invasiveness, self-renewal, epithelial-mesenchymal transition and chemotherapeutic resistance in HNSCC CSCs.[26] Koukourakis et al reported that low ALDH expression is associated with poor prognosis, in comparison to its over expression.[27]

CD24 - It is a mucin adhesion molecule, expressed by pre - B lymphocytes and neutrophils during cell development.[23] It is affiliated with tumor growth and contributes to tumor growth and metastasis.[28]

CD10 - A zinc dependent metalloendoprotease, which is found in normal tissues. It has been affiliated with tumor size, histological grade of malignancy, local recurrence and therapeutic resistance in HNSCC.[29]

c-Met - A tyrosine kinase receptor for hepatocyte growth factor (HGF), has been reportedly linked to tumor invasion, metastasis and decreased survival in HNSCC.[30]

ABCG 2, ABCG 5 - ATP binding cassette (ABC) family including ABCG2, ABCG5 are involved in pumping the Hoechst 33342 DNA dye out of the dye resistant “side population (SP) “ cells. These SP cells are related to drug resistance, self-renewal and tumor initiation.[6] ABCG2 is a potent marker in oral squamous cell carcinoma (OSCC) and is also foreboding of potential cancerous transformation in oral leukoplakia.[31] Increased ABCG5 expression is affiliated with progression and recurrence in OSCC, thus deeming it a potential prognostic marker.[32]

SOX2, OCT4, NANOG - These three transcription factors act as regulators for self-renewal and maintenance of undifferentiated state in embryonic stem cells, thus classifying as stemness markers.[9] SOX2 has been linked to the process of tumorigenesis, and its upregulation has been reported in tumors of squamous lineage.[33] In HNSCC, it is confederated with cell proliferation, migration, invasion, anti-apoptosis, radio chemoresistance and thus, poor prognosis.[34] OCT4 is crucial for epithelial-mesenchymal transition and also has been linked to oncogenic process. In oral SCC, positive expression of this marker is seen in metastatic lymph nodes and recurrent tumors.[35] Moreover, it is associated with poor survival in HNSCC and contemplated as an independent prognostic marker for the same entity.[36] The expression of NANOG is amplified in HNSCC CSCs, and is linked with tumor transformation, tumorigenicity and metastasis. It also corresponds with poor differentiation status, chemoresistance and thus, poor prognosis in HNSCC.[37]

Bmi-1 - It is an epigenetic regulator, a stemness related gene, which maintains the self-renewal ability of stem cells by modulating the chromatin structure.[5] It promotes cell proliferation and is also involved in carcinogenesis.[38] In HNSCC, it is linked with self-renewal, colony formation, migration, invasion and is strongly associated with advanced stages, aggressive clinicopathological behavior, drug resistance and thus, poor prognosis.[39]

Krüppel-like factor 4 (KLF4) - This transcription factor shows an inconsistent pattern in HNSCC, with majority of HNSCC patient samples showing decreased expression. However, HNSCC with increased KLF4 expression is linked with low disease specific survival, while ectopic expression advances tumor progression.[40] Abrigo et al in their study using a mouse model showed that knockout of Klf4 in oral epithelium increases the chances of malignant squamous cell carcinoma (SCC) lesions, hinting towards probable tumor suppression role.[41]

CONCLUSION

CSCs are a small sub-population of cells within a tumor mass capable of tumor initiation and progression. They are also culpable for aggressiveness, recurrences and therapeutic resistance of tumors. Thus, a proper understanding of these cells and its microenvironment would aid in designing better targeted therapeutic strategies for improved clinical outcome. Conventional therapeutic treatment (radiotherapy and chemotherapy) when combined with CSC targeted therapy would show promising results in cancer management.

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Conflicts of interest

There are no conflicts of interest.