Cancer stem cells (CSCs), defined by a small fraction of cells within the bulk tumor have the ability of self-renewal and generating new tumors, are being the hot spots in recent cancer research [6]. It has been considered that CSCs might be responsible for cancers' relapse and metastasis. These features also characterize embryonic stem cells (ESCs), thus suggesting common molecules might exist between CSCs and ESCs [7], [8]. SOX2 (SRY-related HMG-box gene 2), initially reported to be linked strongly with the inhibition of neuronal differentiation, has been shown to acts as an important transcriptional factor to maintain the self-renewal capability of ESCs [9]. OCT4 (OCT3/4), a member of the family of POU domain transcription factor and known to bind in partnership with SOX2, is also the key regulator essential for the pluripotency and self-renewal of of ESCs [10]. Nanog, a homeodomain-containing protein, maintains pluripotency of mouse ESCs by inhibiting NFκB and cooperating with Stat3 [11]. The intermediate filament protein Nestin, initially considered as a marker of neural stem cells, is found to be abundant in ESCs-derived progenitor cells that have the capability to develop into multi-lineages [12]. Of interest, accumulating evidence indicates that these transcription factors of ESCs have a strong correlation with CSCs, knockdown of these genes could decrease tumor sphere formation and inhibit tumor formation in xenograft tumor models [13]–[15]. More importantly, upregulation of these proteins was associated closely with tumor metastasis and poor prognosis in various human malignances including prostate cancer, lung adenocarcinoma, gliomas, rectal cancer, gastric carcinoma and oral squamous cell carcinoma [16]–[21]. To our knowledge, several studies have described the expression of SOX2, OCT4 and Nanog in NPC cells lines [22], [23]. However, up to date, the correlations of these molecules with clinicopathological features and patients survival still remain poorly understood.

Epithelial-mesenchymal transition (EMT) is defined by the loss of epithelial morphology and the acquisition of a mesenchymal phenotype, which is initially found to be central program in early embryonic morphogenesis [24]. In a few years, evidence has mounted for EMT as the key means through which cancer cells acquire more highly mobile potentials to migrate and metastasize to distant sites during tumor progression [25]. E-cadherin, a classical cadherin from the cadherin superfamily, is required for maintaining epithelial cell plasticity. N-cadherin, known as an important member of the cadherin family that mediates calcium-dependent adhesion, is normally expressed in mesenchymal cells. Loss of E-cadherin and increased N-cadherin expression (E/N-cadherin switch) is now defined as a major hallmark of EMT [26], [27]. Snail, one member of the zinc finger family composed of a highly conserved COOH-terminal region, induces EMT and tumor invasion by binding the E-cadherin promoter through E-box sequences. Over the past few years, accumulating data has demonstrated that EMT correlates closely with the acquisition of stem cells-like properties in cancer cells [28], [29]. For example, the Weinberg laboratory reported that the induction of EMT in immortalized human mammary epithelial cells (HMLEs) leaded to the acquisition of stem-like characteristics by using different EMT-inducers including Snail [30]. However, the connection between EMT and stem-like cells in human solid tumor tissues has not been fully described.

Embryonic stem cells (ESCs) are defined as cells that have their ability to self-renew and to differentiate into a variety of adult tissues and cell types. It is generally considered that SOX2, OCT4 and Nanog are key transcription regulators that maintain the pluripotency and self-renewal properties of ESCs [9]–[11]. Growing data demonstrates that stable expression of SOX2, OCT4 and Nanog could promote tumor cell growth, anti-apoptosis and metastasis in vitro and in vivo, therefore play an important role in carcinogenesis [34]–[36]. Of importance, these ESCs-associated proteins were highly expressed in various cancers and contributed to tumor aggressiveness and poor outcome [13]–[15]. However, little is known about the expression levels of these molecules and their correlations with clinical significance in NPC patients. Compared with non-tumoral epithelium, we observed that the expression levels of SOX2, OCT4 and Nanog were highly increased in NPC tissues, respectively, suggesting that these molecules might be involved in the pathogenesis of NPC. Furthermore, our results also revealed that the high expression of SOX2, OCT4 and Nanog was closely associated with tumor aggressive behaviors of NPC patients. For example, both OCT4 and Nanog expression correlated significantly with T classification, N classification and clinical stage. Furthermore, patients with coexpression of OCT4 and Nanog had significantly worse overall survival. Similar to our observations, coexpression of Oct4 and Nanog was found to link significantly with tumor aggressiveness and poor prognosis of several malignances including lung cancer, oral cancer and hepatocellular carcinoma [16], [20], [37]. In fact, a direct link was investigated between OCT4 and Nanog, and they jointly controlled a cascade of pathways to govern the pluripotency and self-renewal characteristics of ESCs [38]. In the present study, a significantly positive relationship between high expression of OCT4 and Nanog was also found. Based on these findings, we suggest that there might be a positive involvement of OCT4/Nanog signaling in tumor invasion and progression of NPC. However, functional impacts of coexpression of OCT4 and Nanog in NPC need to be further examined. Additionally, it is well known that EB-virus infection is strongly associated with NPC carcinogenesis. Therefore, it is of importance to further detect the potential correlation between EBV infection and ESCs-associated biomarkers.

The mesenchymal phenotypic changes by increased motility and invasiveness of epithelial tumor cells are known as the epithelial-mesenchymal transition (EMT) [24], [25], [28]. As a feature of aggressive tumors, EMT is characterized by a switch from E-cadherin to N-cadherin expression, which has been found to correlate with tumor progression and metastasis [26], [27]. Our previous findings also showed that aberrant E/N-cadherin expression contributed to tumor progression and poor outcome of NPC [31], [32]. Of interest, we here showed that overexpression of SOX2, OCT4 and Nanog was significantly associated with high expression of N-cadherin, but adversely with low E-cadherin expression (except for SOX2). Additionally, overexpression of these proteins correlated strongly with the expression of Snail, a central transcription factor as E-cadherin repressor. In line with our observations, several studies in intro reported that overexpression of SOX2, OCT4, and Nanog, individually or simultaneously, leaded to the induction of EMT [16], [39], [40]. Particularly, we found that distributions of SOX2, OCT4 and Nanog staining were more frequently located in the invasive front of tumors, and these cells often exhibited a fibroblast-like, spindle-shaped phenotype. Recently, we have demonstrated that these spindle cells in the invasive tumor front correlated strongly with EMT in NPC tissues [41]. Taken together, our findings strongly indicate that these stem cells-like cancer cells might strongly resemble cells that have undergone an EMT. As expected, high expression of these proteins in the invasive front correlated significantly with a majority of tumor aggressive behaviors in NPC, such as tumor infiltration, lymph node metastasis and distant metastasis. To the best of our knowledge, no reports have previously described the prognostic impacts of these ESCs proteins in the invasive front of tumors. Collectively, we speculate that tumor cells with stem-like properties in the invasive tumor front of NPC are capable of generating tumor invasion and metastases.

The niche concept was firstly defined as “stem cell niche”, which is composed of diverse stromal cells including mesenchymal and immune cells, and regulates self-renewal, proliferation, and apoptosis resistance of stem cells [42], [43]. Like normal stem cells, it is currently thought that CSCs also rely on a “CSC niche”, to maintain their exclusive abilities to self-renew and grow more differentiated cells [44]–[46]. Of note, vascular niche has recently proved to be responsible for the induction of CSCs properties [47]. For example, Calabrese et al. demonstrated that brain tumor CSCs might live in a “vascular niche” that stimulates their self-renewal. Disrupting this niche impaired CSC self-renewal and significantly inhibited tumor growth [48]. Nestin, is an intermediate filament protein known as a stem/progenitor cell marker, which is normally expressed in undifferentiated central nervous system (CNS) cells, but also in endothelial cells [49]. More recently, He H et al. reported that Nestin-positive blood vessels were crucial for maintaining the structure of the glioma stem cell niche [50]. Interestingly, in the present study, Nestin expression was predominately found in endothelial cells at the invasive front of NPC. Furthermore, tumor cells exhibiting CSCs-like features correlated significantly with Nestin staining in the invasive front. Based on these findings, we postulate that vascular endothelial cells expressing Nestin might represent the CSCs niche in NPC. Surely, functional investigations are warranted to further elucidate this hypothesis.

Table S1