CURRENT CANCER STEM CELL BIOMARKERS IN TONGUE SQUAMOUS CELL CARCINOMA

Tongue squamous cell carcinoma (TSCC) is known to be the most malignant cancer type amongst other oral cancers with increasing incidence and mortality rates in the past five years. Since the life expectancy for TSCC patients is limited and the current chemo-radiotherapy treatments are not curative, novel biomarkers are urgently needed. As many other solid tumors, TSCC has a heterogeneous cancer cell population, which includes a small subpopulation identified as cancer stem cells (CSCs) that are considered as the driving force for tumor initiation, development, spread, recurrence, and resistance to chemoradiotherapy. Although the underlying molecular mechanisms of how CSCs are involved in the carcinogenesis are not completely understood, scientists and clinicians aim to utilize those cells as therapeutic tools in fight against different cancer types including TSCC. Here, we reviewed and summarized important findings and the most current literature to shed light on the potential of cancer stem cells markers in TSCC. Possible functions of CSCs biomarkers in TSCC pathogenesis during cancer initiation, progression, invasion or metastasis are also summarized.


Introduction
Oral cancer is the sixth most frequent cancer type worldwide with an annual global incidence of 300.000 cases and 130.000 deaths (Brockton et & Li 2016).Tongue squamous cell carcinoma (TSCC) is a very aggressive disease and is the most common cancer type of the oral cavity making up almost 40% of all oral carcinomas (Cui et al. 2017).TSCC is also known to be the most malignant cancer type amongst other oral squamous cell carcinomas and causes speech, mastication and deglutition problems (J.Chen & Li 2017, Zhang et al. 2017).Although overall cancer incidence and mortality rates reduced in the past five years, incidence and mortality rates of TSCC increased significantly within the same period (Siegel et al. 2016).In addition, contrary to improvements in multimodal diagnosis and treatment techniques in the past few decades, the 5-year survival rate for TSCC stayed almost unchanged, which makes it one of the most lethal cancer types seen in head and neck region (Xing et al. 2013).Therefore, novel early diagnostic and prognostic markers are urgently needed for TSCC since the life expectancy for patients is very limited and the current chemo-radiotherapy treatments mostly fail to give positive clinical outcomes (Alam et al. 2017Boldrup et al. 2015).
As in many other solid tumors, TSCC has a heterogeneous cancer cell population (Weng et al. 2017) which includes a small subpopulation identified as cancer stem cells (CSCs).These stem cells are thought to be responsible for tumor initiation, development, spread, recurrence, and resistance to chemo-radiotherapy.Therefore, their identification, isolation, and characterization are of paramount importance to enhance the success of treatment of TSCC through development of CSCs targeted therapy techniques.
Here, we reviewed and summarized important findings and the most current literature to shed light on the potential CSCs markers in TSCC.Possible functions of these CSCs biomarkers in TSCC pathogenesis during cancer initiation, progression, invasion or metastasis were summarized.

Presence of CSCs in TSCC
CSCs were initially identified in leukemia through isolation of a subpopulation with tumor initiating potential, which suggested that tumors are comprised of hierarchically organized heterogeneous cell populations (Vlashi & Pajonk 2015).Further studies conducted with pediatric brain tumors demonstrated the presence of CSCs for the first time in solid tumors (Hemmati et al. 2003).
CSCs were shown to have essentially similar properties as normal stem cells in terms of their selfrenewal, unlimited proliferation, and differentiation potentials.CSCs and normal stem cells utilize similar signaling pathways (Dawood et al. 2014)  CSCs are often the main source of genetic abnormalities for malignant transformation (Li et al. 2015) and unlike normal stem cells, they initiate and propagate tumor when they are transplanted into immunodeficient model organisms and take an active role in metastasis, recurrence, and resistance of tumor to chemo-radiotherapy.As a result of these features, CSCs are referred as "tumor-initiating cells" or "tumorigenic cells".Surface markers such as CD133, CD24 and CD44 are typically used for isolation and identification of CSCs (Dawood et al. 2014).
In the last decade, researchers demonstrated the presence of CSCs in TSCC through isolation and characterization of specific cell populations expressing certain surface markers such as CD133 and CD44 (Sun et al. 2012).CD133 and CD44 expressing cells were demonstrated to have increased tumorigenic potential both in vitro and in vivo (Saleem et al. 2014, Wang et al. 2016).Another recent study identified two different cancer stem cell subpopulations, p63+/NANOG+/SOX2+/SALL4+/pSTAT3+/OCT4-and p63-/NANOG-/SOX2-/SALL4-/pSTAT3-/OCT4+ in moderately differentiated TSCC (Baillie et al. 2016).Besides, ALDH1 positive cells exhibited increased proliferation and differentiation capacity compared to ALDH1 negative cells in Tca8113 cell line, which confirmed the presence of cell clusters showing CSCs properties in human TSCC (Zou et al. 2012).
However, further efforts are needed for elucidation of genetic and epigenetic circuits modulating the stem cell features of CSCs to help understanding the molecular basis of carcinogenesis.

CD44
CD44 and its variants are among the well-described and studied biomarkers in TSCC (Kunishi et al. 1997, Sato et al. 2000).CD44, located on chromosome 11, encodes for a conserved transmembrane glycoprotein which is necessary for the maintenance of tissue integrity.It has been shown to be deregulated in several cancers including TSCC (Lindquist et al. 2012, Yanamoto et al. 2014).CD44 has important roles during carcinogenesis and metastasis in TSCC and is involved in several carcinogenic processes like proliferation, migration, cellcell interactions, and apoptosis in TSCC cells ( The expression of CD44 in TSCC samples was initially investigated in 1996, where 83 head and neck squamous cell carcinoma (including 12 TSCC specimens) samples were used for immunohistochemical analysis of CD44 variants (v5, v6, v7, v7-v8, and v10) in normal, dysplastic squamous epithelia, primary and metastatic squamous cell carcinoma samples.CD44 v7, v8, and v10 were reported to be significantly downregulated in primary tumor tissues and were not detectable in most of the metastasis-derived specimens.On the other hand, v5 and v6 of CD44 displayed no significant alteration between normal and tumor samples (Herold-Mende et al. 1996).However, in the study of Kunishi (1997), expression level of CD44v6 was found to be significantly reduced in tumor samples when compared to normal healthy mucosa.Decreased expression of CD44v6 was also associated with regional lymph node metastasis.Interestingly, although no significant correlation was found between the expression of CD44v6 and level of differentiation, poorly differentiated carcinoma samples tended to express reduced level of CD44v6 (Kunishi et al. 1997).In the following years, reduced expression of different CD44 variants was associated with different clinical parameters such as CD44H with late nodal metastases following interstitial brachytherapy (Masuda et al. 2000), CD44v9 with lymph node metastasis and poor survival of patients (Sato et al. 2000), CD44 v3, v4-5, and v6 with cell differentiation, tumor grade, and the pattern of neoplastic invasion (Fonseca et al. 2001), CD44v6 with high histological grade (Cruz et al. 2009) and CD44 with cervical lymph node metastasis (Mostaan et al. 2011).CD44 expression was also demonstrated to be profoundly reduced in TSCC compared to other tumors within the oral cavity (Krump & Ehrmann 2013).Furthermore, loss of CD44 expression in normal epithelia of tongue was suggested as an early biomarker for tongue carcinogenesis (González- Moles et al. 2004) and its expression was demonstrated to be strongly reduced in tongue tumors in comparison to other sub-sites within the oral cavity (Krump & Ehrmann 2013).
The first findings about the elevated expression of CD44 in TSCC were obtained in the study of Järvinen et al. (2008) where the authors characterized genome-wide copy number and gene expression alterations on microarrays for 18 TSCC cell lines.They identified several high-level amplifications including 11p12-p13 region where CD44 gene is localized.In addition, high intensity CD44 staining was demonstrated as a strong indicator of poor prognosis (Lindquist et al. 2012) and was significantly associated with regional lymph node metastasis, pattern of invasion, depth of invasion, perineural invasion and local recurrence (Yanamoto et al. 2014).
CSCs were reported to be enriched in the highly invasive UM1 cell line which is strongly positive for CD44 expression but were not present in the less invasive UM2 cell line (Misuno et al. 2013).In a recent study, where 4-Nitroquinoline 1-oxide was used to induce tongue cancer in mice, CD44 expression along with CD133 was found to be slightly overexpressed in dysplasia group compared to normal rats (Lim et al. 2014).Furthermore, CD44 and CD133 expressions were found to be strongly overexpressed in 4-Nitroquinoline 1oxide induced TSCC in addition to several other cancer stem cell markers including ALDH1, Nanog and OCT-4.These findings point out the importance of CD44 as well as other cancer stem cell markers in the multistep carcinogenesis process of TSCC (Lim et al. 2014).
In another recent study, a cell line called UM-SCC-103 was developed by isolating highly aggressive tumor cells from a pregnant woman diagnosed with TSCC.Sorted CD44+ cells from UM-SCC-103 were able to induce carcinogenesis, whereas, CD44-cells failed to produce the overall heterogeneity of the primary tumor.This study suggested that CD44 is an important putative marker for tongue CSCs (Owen et al. 2014).

Saleem et al. (2014) used biopsy specimens from
TSCC and its neck nodules for development of primary cell cultures and examined the growth and sphere formation capacity of those cells.They found the lowest CD44 expression in hyperplastic tongue tissue and the highest expression in neck node positive TSCC specimens.Cells with increased CD44 expression showed much higher sphere-forming capabilities compared to the corresponding CD44-cells.Interestingly, CD44+ cells from hyperplastic or non-cancer tongue tissue did not produce spheres.Besides, primary cultures from metastatic TSCC specimens showed stronger CD44/CD24 expression and produced much more spheres in significantly shorter duration compared to those of node negative TSCC and normal tongue specimens (Saleem et al. 2014).
Besides, injection of CD44+ sorted and labeled TSCC CSCs into the tongue of nude mice resulted in formation of highly metastatic tumors which were larger in size compared to CD44-and unsorted SCC-9 cells.Tumor formation efficiency was 100% in CD44+ group, but CD44-group showed slightly reduced tumor formation efficiency.Wu et al. (2017) also demonstrated that CD44 expression in human clinical samples was significantly higher in metastatic tumors than in primary tumor samples.
The evidence presented here demonstrates that CD44 expression has the potential to suppress and induce cancer progression.Discrepancies reported in the literature might stem partially from differences in the cell lines used, antibody variability, culture conditions, and other experimental variability.However, the conflicting data about the potential of CD44 as a stem cell marker needs further studies and clarification for its successful utilization in therapeutic approaches against TSCC.

CD133
Cluster of differentiation (CD) 133 (also known as AC133; human homologue of mouse Prominin-1) is a highly conserved pentaspan transmembrane glycoprotein encoded by PROM1 on chromosome 4 (4p15.32)(Yanagisawa et al. 2005).It consists of 865 amino acids and has a total weight of 120 kDa.Its expression has been associated especially with progenitor/stem cells and differentiation (Li 2013).Subsequently, CD133 has been identified as a promising CSCs surface marker in several cancer types including larynx, lung, brain, skin, colon carcinoma and TSCC (Li 2013 CD133+ cells were initially identified in TSCC Tca8113 cell line by Kang et al. who showed that a small portion of Tca8113 cell line were positive for CD133, and these CD133+ cells represented high proliferation capacity compared to corresponding CD133 negative (CD133-) control Tca8113 cells (Kang et al. 2010).This study pointed the importance of CD133 as a putative CSCs surface marker for tumor-initiating cells of human TSCC (Kang et al. 2010).In another study carried out with Tca-8113 cell line, it has been shown that proliferative capacity and differentiation potential of CD133+ cells were profoundly higher than those of CD133-cells in vitro (Wang et al. 2016).CD133+ cells were also characterized with their relatively strong tumorigenic potential in vivo (Wang et al. 2016).In a 4-Nitroquinoline 1-oxide-induced rat tongue carcinogenesis model, CD133 expression during the progress of multistep carcinogenesis was slightly increased in the dysplasia group compared with normal rats, but the expression level was significantly increased in rats with TSCC along with other important CSCs markers.This study proposed CD133 as a crucial CSCs surface marker for identification of CSCs with a high potential of oral carcinogenesis (Lim et al. 2014).
In a recent clinical study conducted by Mascolo et al. (2012) the expression level of CD133 in the oral squamous cell carcinoma samples mostly comprised of TSCC was significantly higher compared to normal oral mucosa samples.
Taking all these findings into account, CD133 has been suggested to serve as an important CSCs surface marker for isolation, identification and characterization of CSCs in TSCC patients, which might help development of novel targeted therapeutic approaches to enhance the quality of life and survival of patients.

SOX2 and OCT4
Qiao et al. (2014) investigated the expression status of stemness markers SOX2 and OCT4 in the tumorigenesis process of oral mucosa by immunohistochemistry using both rat and human samples.They demonstrated that individual expression and co-expressions of SOX2 and OCT4 were detected in normal oral mucosa, premalignant diseases, primary and metastatic sites of oral squamous cell carcinoma.However, co-expression of SOX2 and OCT4 in transforming oral mucosa cells were suggested to contribute to the malignant transformation of oral mucosa (Qiao et al. 2014).In another study performed in 2016, lentiviral transduction of Sox2 and Oct4 together into immortalized oral epithelial cells triggered formation of neoplasms in immunodeficient mice, but their individual introduction into immortalized oral epithelial cells did not result in tumor formation.Furthermore, injection of Cal27 cells, with simultaneous knockdown of Sox2 and Oct4, into immunodeficient mice caused reduced tumor size.Besides, positive expressions of OCT4 and SOX2 were preferentially located in the nucleus of tumor cells obtained from 51 TSCC patients (Jiang et al. 2017).These findings strongly indicate that both SOX2 and OCT4 might be required for reprogramming of cancer stem cells for induction of oral carcinogenesis (Cai et al. 2016).
Quantitative proteomic analysis of sphere-forming CSCs of highly invasive UM1 and low invasive UM2 cells both obtained from the same tongue squamous cell carcinoma patient demonstrated that UM2 cells did not possess sphere-forming CSCs.On the other hand, sphereforming CSCs of UM1 cells were strongly positive for several stem cell factors including SOX2 and OCT4.Along with those markers, many proteins in cell cycle, metabolism, G protein signal transduction, translational elongation, development, and RNA splicing pathways were differentially expressed among the two cell phenotypes, which might be associated with elevated levels of stem cell factors like SOX2 and OCT4 (Misuno et al. 2013).

KLF4
Interestingly, another reprogramming factor, KLF4, has been found to play Janus-faced roles in oral cancer carcinogenesis, acting both as a tumor suppressor and as an oncogene.The expression of KLF4 was found to be lower in the poor-differentiated oral cancers compared to the well-differentiated cancers.KLF4 functions as a tumor suppressor in vitro and/or in vivo through suppressing cell proliferation, cell cycle progression, cell colony formation and by inducing apoptosis (W.Li et al. 2015).It was also shown to inhibit cellular proliferation and induce differentiation to help maintenance of epithelial homeostasis.In a recent study in which an inducible oral-specific mice model was utilized to knockout Klf4 specifically in the oral cavity, dysplastic lesions with increased cell proliferation and abnormal differentiation were reported in the tongue cells four months after induction (Abrigo et al. 2014).Utilization of 4-nitroquinoline 1-oxide along with Klf4 knockout in the oral cavity resulted in development of more severe dysplastic lesions in the oral cavity and a tendency for increased incidence of oral squamous cell carcinoma (Paparella et al. 2015).These findings support the potential of KLF4 as a tumor suppressor for TSCC, however, upregulation of KLF4 induced TSCC cell migration and invasion in vitro.In addition, although knockdown of KLF4 in TSCC cells increased cellular proliferation and colony formation, it significantly resulted in inhibition of cell migration and invasion.Contradictory to its tumor suppressor function, these recent findings assign an oncogenic role for KLF4 in TSCC.Therefore, further studies are necessary to clarify the roles of KLF4 as a CSCs marker during tongue carcinogenesis process.

NANOG
Overexpression of Nanog has been previously reported in several types of tumors including oral squamous cell carcinoma (Bourguignon et  been suggested to play critical roles during carcinogenesis, tumor-progression, and metastasis (Ratajczak et al. 2007, Trosko 2006).Nanog expression was found to be significantly higher in tongue tumor tissues than in corresponding normal oral mucosa tissues (Fu et al. 2016).Nanog expression together with ALDH1 and OCT4, which are other CSCs markers, were reported to significantly increase during multistep carcinogenesis in a 4-nitroquinoline 1-oxide-induced rat tongue carcinogenesis model (Lim et al. 2014).Besides, longterm exposure of the carcinogenic 4-methylnitrosamino-1-3-pyridyl-1-butanone resulted in promoted migration and invasion in a dose-dependent manner induced overexpression of several CSCs markers including Nanog (Nieh et al. 2015).Nanog expression together with Oct3/4 were also reported to be significantly upregulated in side population compared to remaining cell population and their expressions were found to be strongly correlated with development of delayed neck metastasis (Habu et al. 2015).Taking into account these findings, Nanog might be considered amongst important CSCs biomarkers for TSCC to be utilized for identification and characterization of CSCs present in TSCC tissues.

BMI1
Bmi1, which plays critical roles in the functioning and maintenance of both endogenous stem cells and CSCs (Tanaka et al. 2013), was demonstrated to be significantly overexpressed during tongue carcinogenesis starting from very early stages (He et al. 2015).A recent study reported that knockdown of Bmi1 in TSCC cell lines stimulated cell apoptosis and senescence along with suppression of cell proliferation and migration.Interestingly, its inhibition also reduced the colony formation efficiency and the proportion of CD44+/CD133+ sub-population with CSCs features (Li et al. 2014).Besides, Bmi1 positive (Bmi1+) lingual epithelial stem cells were reported as the origin for tongue cancer in 4nitroquinoline-1-oxide induced tongue cancer models since certain Bmi1+ cells were found to proliferate constantly, which eventually resulted in the formation of tumors derived from single Bmi1+ cells.This in vivo model provided invaluable data about the presence of CSCs within TSCC specimens and pointed out the importance of Bmi1 as a critical CSCs marker (Tanaka et al. 2016).

NOTCH
A recent meta-analysis study explored the role of Notch signaling pathway in human oral squamous cell carcinoma to understand the role of Notch signaling in during oral carcinogenesis (Osathanon et al. 2016).The researchers utilized 13 Gene Expression Omnibus datasets including those carried out with TSCC samples and found that oral squamous cell carcinoma specimens showed significant overexpression of several genes in Notch signaling pathway including JAG1, JAG2, ADAM17, NCSTN, PSEN1, NCOR2, NUMB, DVL3, HDAC1, and HDAC2, pointing out the importance of Notch and its effectors for TSCC carcinogenesis (Osathanon et al. 2016).Notch is reported to be amplified and overexpressed in significant proportion of early stage TSCC tissue samples.Overexpression of Notch, which is characterized as inducer of stem cell maintenance in potential cancer-initiating cells, in TSCC cell lines caused enrichment of stem cell markers and formation of spheroids with CSCs features.Conversely, its inhibition suppressed spheroid forming capacity, transformation, survival and migration of TSCC cells, indicating an oncogenic role for Notch in TSCC through regulating stem cell characteristics (Upadhyay et al. 2016).

EMT Markers
Accumulating evidence shows that epithelial mesenchymal transition (EMT), which is a significant physiological process where epithelial features are lost and mesenchymal properties are acquired (Iwatsuki et al. 2010, Thiery et al. 2009), is a crucial process playing critical roles during embryogenesis and carcinogenesis (Vered et al. 2010).It has been recently proposed that CSCs undergo an EMT for acquisition of migratory and invasive phenotype, which is necessary for progression of tumor (Pan et al. 2016).
In a recent study, treatment of Tca-8113 cells with Bone morphogenic protein 4 (BMP-4) was reported to promote EMT through acquisition of stem cell associated phenotypes (Qiao et al. 2011).BMP-4 significantly upregulated mesenchymal markers like Snail, Slug and Vimentin, and downregulated the epithelial marker Ecadherin along with overexpression of stemness gene which was not synchronous with the expression of EMT markers (Qiao et al. 2011).Another study investigating the expression profile of Bmi1, which is involved in selfrenewal of stem cells, and that of ZEB1, a transcription factor that is associated with EMT, in TSCC cells and tongue specimens revealed that Bmi1 and ZEB1, both in mRNA and protein levels, were observed at the invasive front of TSCC cells, which were accompanied by the downregulation of epithelial markers and overexpression of mesenchymal markers both in vitro and in vivo (Kurihara et al. 2015).In addition, induced expression of Snail and Slug, two important EMT markers, resulted in a mesenchymal phenotype and morphology and induced cell invasion and stem cell related features in TSCC cells (Zheng et al. 2015).

Biomarkers in TSCC Prognosis
In addition to their tumor initiating and promoting properties, CSCs are significantly associated with cancer progression and clinical outcome and they are thought to be responsible for tumor development, metastasis, and recurrence as well as tumor initiation (Guzel et al. 2014).In addition, since CSCs and normal tissue stem cells share many similarities and CSCs are significantly involved in the malignant progress of tumors (Table 1), they are considered among the critical targets for future therapies.The expressions of ALDH1, CD44, OCT4 and SOX2, as important cancer stem cell markers, were investigated in a recent clinical study by immunohistochemistry utilizing 66 TSCC tissue specimens (Huang et al. 2014).This study demonstrated that all of these CSCs markers were significantly overexpressed in TSCC samples, however, only the expression of SOX2 along with recurrence and distant metastasis were found as individual independent prognostic factors of overall survival in TSCC patients (Huang et al. 2014).Moreover, in another study, where tumor tissue samples from 82 histologically node-negative TSCC patients were evaluated with immunohistochemistry, SOX2 expression was significantly associated with large tumor size, and its increased expression was related with poorer overall, cancer-specific and disease-free survival of patients (Du et al. 2011).In a different study, paraffin embedded tissue specimens of 51 patients with TSCC were examined immunohistochemically, and OCT4 and SOX2 expressions were found to be significantly associated with histological grade of TSCC and poor overall survival of patients (Jiang et al. 2017).Moreover, OCT4 expression, but not SOX2, was also significantly correlated with lymph node metastasis.In the meantime, SOX2 was suggested as an independent prognostic factor for overall survival (Jiang et al. 2017).In addition, along with vascular invasion, expression of OCT4 was reported to be a potential predictor for identification of patients with high risk of delayed neck metastasis (Habu et al. 2015).Taking these findings into account, certain CSCs markers including SOX2 and OCT4 might be proposed as independent prognostic factors for patient survival in TSCC.
Apart from SOX2 and OCT4, deregulation of another important stem cell marker, Bmi1, was demonstrated to be a common event during TSCC progression.Its overexpression was significantly associated with poor survival of TSCC patients.Late stage patients and patients with a positive node metastasis tended to have significantly higher Bmi1 expression compared to early stage patients and patients with a negative node metastasis, respectively (He et al. 2015).Its overexpression was also reported in a major fraction of tumor samples obtained from 52 patients with no prior history of chemotherapy or radiotherapy, and the overexpression was significantly associated with cervical node metastasis and reduced overall survival, revealing Bmi1 as an independent prognostic marker for TSCC patients (Li et al. 2014).On the other hand, an earlier study showed that negative or reduced Bmi1 expression was related to recurrence of patients and suggested negative Bmi1 expression as a biomarker for poor prognosis in TSCC patients (Häyry et al. 2010).The contradictory findings of the present studies question the power of Bmi1 as a prognostic marker and require further investigations to enlighten its roles in the progression of TSCC.
In several studies, reduced expression of different isoforms of CD44, as a widely investigated CSCs biomarkers in TSCC, was reported to be correlated with different clinical parameters including regional lymph node metastasis (Kunishi et al. 1997) Yu et al. 2015).Thus, the exact role of CD44 for prediction of TSCC progression is not clear yet and further efforts are needed for clarification of its power for estimation of patients' prognosis in TSCC.
Expression of CD147, a potential oncogenic CSCs marker in TSCC, was significantly correlated with tumor diameter, clinical stages of the TSCC tumor samples and poor overall patient survival (Huang et al. 2009).CD147 expression level was also demonstrated to have significant association with poor prognosis, a higher T stage (Yu et al. 2015), recurrence, and node metastasis (Huang et al. 2012).

Future Potential
Experiencing a recurrence after a successful therapy is quite frequent in various cancer types including TSCC.Besides, current conventional treatment options mostly fail to result in a positive clinical outcome in advanced cases, which ultimately results in cancer related death due to disease progression and related organ failure.Recent studies indicated that CSCs are critical contributors of tumor initiation, progression, metastasis, recurrence, and chemo-radioresistance.Although underlying molecular mechanisms of how CSCs contribute to cancer pathogenesis is not completely clarified and understood, scientists and clinicians aim to use those cells as therapeutic tools to fight against cancer.Unraveling both genetic and epigenetic machineries of CSCs is necessary to develop effective and successful therapeutic options.
One of the major challenges of CSCs research is the accurate identification and characterization of CSCs, which is needed for their specific targeting.Therefore, to develop a therapeutic approach aiming at preferentially killing CSCs, the initial goal needs to be the identification of true surface marker(s) and stemness genes in CSCs.To do so, further development of anti-CSCs agents will help overcoming the tumor chemoresistance or radioresistance.Furthermore, early detection and accurate diagnosis are especially crucial for determination and application of effective cancer therapy options in the clinical decision making process.Detailed characterization of circulating tumor cells, especially CSCs, will give opportunity to predict the prognosis of the cancer and decide the personalized therapy strategies for each patient.
In conclusion, the area of CSCs research is in its infancy and current understanding of the CSCs biomarkers in tongue pathogenesis is quite limited.Therefore, further detailed investigations are essential for better understanding of the roles and functions of CSCs biomarkers in TSCC biology.
and both have the ability to activate anti-apoptotic signals and have increased telomerase activity (D'Arcangelo et al. 2015).
called 'the Yamanaka Factors' have been shown to play critical roles in acquisition and maintenance of self-renewal and pluripotency characteristics in embryonic stem cells via their interaction with other transcription factors as well as important signaling molecules (Boyer et al. 2005, Loh et al. 2006, Patel et al. 2014).Interestingly, the gene expression profiling of CSCs and the rest of the tumor burden pointed the differential expression of those stemness-associated genes in CSCs (Chen 2009, Zhang et al. 2012).

Table 1 .
TSCC CSCs biomarkers associated with prognosis of disease.
(Krump & Ehrmann 2013,s(Masuda et al. 2000), cervical lymph node metastasis(Mostaan et al. 2011), poor survival of patients(Sato et al. 2000), 5-year disease-free survival(Dunkel et al. 2013), and high histological grade(Cruz et al. 2009).However, increased expression of CD44 in TSCC samples, in accordance with its potential for contribution to CSCs features, was also associated with poor prognosis(Lindquist et al. 2012) and tumor local recurrence(Yanamoto et al. 2014).In the meanwhile, there are reports showing no statistically significant association between the expression of CD44 and different clinical or histological parameters(Krump & Ehrmann 2013,