Stemness state regulators SALL4 and SOX2 are involved in progression and invasiveness of esophageal squamous cell carcinoma

Normal and CSCs show similarities in different aspects such as gene expression profile, activated cell signaling pathways and epigenetic modifications which play important roles in cell fate decision. CSCs, as a subgroup of tumor cells, contain specific mechanisms and molecular networks contributed to cancer initiation and progression, as well as therapeutic resistance. Having established a tumor, CSCs are able to sustain tumor mass growth and development leading to metastasis of the cells [1]. Therefore, it is tempting to speculate the existence of master regulatory transcriptional network of ESCs consisting of (but not limited to) SALL4 and SOX2 in CSCs. Our results may confirm such extrapolation for ESCC.

Here we demonstrated the significant overexpression of SALL4 and SOX2 in ESCC tissues and illustrated significant correlation between the expressions of these genes. Furthermore, we revealed the clinical relevance of SALL4 and SOX2 in ESCC and demonstrated that high levels of both genes’ expression were associated with progression and lymph node metastasis of tumor cells, suggesting that SALL4/SOX2 transcription factors play important roles in the beginning and continuing of ESCC invasion and metastasis.

SALL4, a member of the SALL (Spalt-like) family of zinc finger transcription factors, is homologue of homeotic gene (Spalt) of Drosophila which is essential for development [3]. It contains multiple C2H2 zinc finger motifs, which are evenly distributed over the protein. SALL4 is a stemness factor which not only plays important roles in embryonic development, maintaining pluripotency and self-renewal state of ESCs, but also elicits reprogramming of somatic cells and determines cell fate decision [5]. SALL4 has a direct interaction with key molecules of cell signaling pathways such as Wnt/β-catenin and BMI-1 to maintain the pluripotency of ESCs during embryogenesis [5].

SOX2 is a master regulator of pluripotency and self-renewal in ESCs and a variety of adult stem cells. This protein is a member of evolutionarily conserved stem cell transcription factors containing a SRY-related HMG box. In addition, SOX2 is known as lineage-survival oncogene which is essential for the expression signature of pluripotent stem cell (PSCs) and the lineage-specific genes in lung squamous cell carcinoma (SCC). Ectopic expression of SOX2 in normal trachea bronchial epithelial cells generates the oncogenic transformation of the cells [22]. Recent studies suggest the oncogenic roles of SOX2 in the organization of SCC in several organs such as esophagus, where it is principally expressed in the basal progenitor cells of the stratified epithelium [23]. It is also expressed mainly in other types of poorly differentiated and aggressive human cancers [24].

Using proteomics screening of SOX2-associated proteins, Mallanna et al. demonstrated that SOX2 interacts with more than 60 nuclear proteins during the early stages of ESCs development. In addition, they showed that SOX2-associated proteins have interactions with other pluripotency factors including SALL4, NANOG, OCT4 and ESRRB in ESCs and specifically identified the interaction of SALL4 with nearly 12 % of SOX2-associated proteins [25]. These finding illustrate the involvement of a complex regulatory network consisting of master proteins such as SOX2 and SALL4 in maintenance of self-renewal and stemness state of ESCs. This regulatory network may be present in CSCs as well. Since our results showed co-overexpression of SALL4 and SOX2 in ESCC and also their significant correlation, we can extrapolate the existence of similar regulatory network in ESCC and its significant involvement in maintenance and progression of the disease through direct or indirect mechanisms in both mRNA and protein levels.

It has been shown that SOX2 overexpression is critical for survival and migration of epithelial cells in lung SCC [26]. SOX2 overexpression has been reported in several solid tumors. The elevated expression of SOX2 was indicated in 95 and 90 % of ESCCs and lung cancers, respectively, where this stemness marker is suggested to be involved in early stages of both cancers’ tumorigenesis [27, 28]. Aberrant expression of SOX2 is identified in significant correlation with the invasiveness state of tumor cells. SOX2 overexpression is correlated with highly invasive and metastatic colorectal cancers and gliomas, while its knockdown decreases aggressiveness of melanomas, gliomas and colorectal tumor cells [13, 18, 19]. It has been reported that SOX2 silencing can induce mesenchymal–epithelial transition (MET) in colorectal cancer cells and causes a significant decline in cell mobility and invasion in vitro and metastasis in vivo [18]. Furthermore, it has been shown that SOX2 overexpression can deregulate cell cycle in breast cancer cell line (MCF7) leading to enhanced cell proliferation and tumorigenesis [29].

Recent studies demonstrated that SALL4 is a metastatic marker for colorectal, endometrial and gastric cancers, and co-overexpression of SALL4 and SOX2 genes plays essential roles in metastasis of laryngeal SCC, breast and liver malignancies. In line with these studies, here we also elucidated the metastatic role of SALL4 and SOX2 in ESCC and introduced these markers as important factors contributed to the invasion and aggressiveness of the disease.

SOX2 and SALL4 are vital regulators of maintaining the self-renewal and pluripotency in ESCs, and their deregulation leads to ESCs differentiation. On the other hand, these proteins can promote epigenetic reprogramming in somatic cells and induce PSC state [30]. In this study, we indicated that expression of SOX2 is associated with SALL4. This investigation is intriguing. Recent studies have showed that the interaction of SALL4 and NANOG can be autoregulated through feed forward loops to maintain their expression in the cell [31]. This connection is similar to the SOX2/OCT4 interaction that was indicated in murine ES cells. Furthermore, it was detected that SALL4/NANOG can bind to the upstream regulatory sequences of OCT4 and SOX2 genes [31]. Therefore, there are autoregulatory and feed forward connections between such master transcriptional factors in ESCs [31]. Similarly, these connections may be found in CSCs. The correlation between SALL4 and SOX2 in ESCC may confirm such connections leading to hypothesize not only the regulatory role of SALL4 on SOX2 gene expression, but also the coordination of these factors to express other pluripotency genes in the tumor cells (Fig. 3a).

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Genome-wide DNA-binding analysis explained that SOX2, OCT4 and NANOG, form a core transcriptional complex which bind to a large group of genes which code for SOX2-associated proteins in ESCs [29]. Furthermore, it indicated the existence of regulatory transcriptional networks which is controlled by SALL4 and eventually led to controlling of the ESCs pluripotency [31]. Interestingly, it has been shown that SALL4, BRG1 and HDAC1 are associated with the promoter regions of the genes which are activated by regulatory complex consisting of S0X2, OCT4 and NANOG in ESCs [31, 32] and are involving in the pluripotency and self-renewal of ESCs [25]. Such interaction between SALL4 and SOX2 in ESCs may be mimicked in CSCs as well to maintain self-renewing of the cells in tumor mass. Co-overexpression of these genes in ESCC and their significant clinicopathological relevance may confirm crucial importance of this regulatory network in ESCC development (Fig. 3a).

It has been detected that SALL4 overexpression is associated with BMI-1 promoter H3–K4 and H3–K79 histone methylation in leukemic stem cells, providing a link between SALL4 and polycomb group proteins in regulation of normal and CSCs self-renewal [5]. Since BMI-1 expression declines with differentiation, SALL4-mediated BMI-1 gene expression through epigenetic mechanism may inhibit cell differentiation. BMI-1, polycomb ring finger oncogene, is a direct target of SALL4 gene which regulate expression of cell cycle inhibitors such as p16 and p19. He et al. [33] have indicated that BMI-1 is overexpressed in early stages of ESCC and inversely correlated with the well-differentiated state of tumor cells, leading to ESCC progression. Such data may emphasize the existence of similar connection between SALL4 and BMI-1 in esophageal CSCs self-renewing and ESCC progression (Fig. 3b).

SALL4 and SOX2 are able to interact with the WNT/β-catenin pathway components in HSCs/CML and breast cancer, respectively. Chen et al. [29] reported that SOX2 protein maintains its function of transcriptional regulation through interacting with β-catenin in breast cancer cells. On the other hand, blocking of canonical WNT/β-catenin signaling inhibits SOX2 expression in retina development, which leads to reduced cell proliferation [34]. Based on these evidences, it is clear that SALL4 and SOX2 may coordinate through WNT/β-catenin pathway. Interestingly, we have previously reported high expression of PYGO2, the main transcription factor of WNT signaling pathway transcriptional machinery, in ESCC in correlation with advanced stages and tumor invasion of the disease [35]. Having combined such in line observations, we may hypothesize the probable interactions between SALL4/SOX2 and WNT pathway in ESCC which can enhance carcinogenesis and aggressiveness of the disease (Fig. 3c).

There is a close relationship between SALL4 and implicated master transcription factors in molecular regulatory network which maintain ESCs pluripotency, including SOX2, OCT3/4, KLF4, c-MYC and NANOG [5]. The coordination between SALL4 and these critical transcription factors can induce reprogramming of differentiated cells to a stemness state of iPS cells, while, down-regulation of SALL4 causes down-regulation of all these genes during somatic cell reprogramming process [5]. Recent studies clearly demonstrated that SALL4 can enhance somatic cell reprogramming by recruiting the stemness state regulatory transcriptional network consisting of SOX2, OCT3/4, KLF4 and c-MYC reprogramming factors [5]. Such observations suggest that SALL4 and SOX2 play a vital role not only in regulation of ESCs self-renewal and pluripotency, but also in reprogramming process of different type of differentiated cells through interaction with core transcription factors of multiple stem cell signaling pathways. Our results may confirm such suggestion for ESCC. Co-overexpression of the genes in ESCC was significantly correlated with the different behaviors of the tumor cells including stage of tumor progression, depth of tumor invasion, metastasis of tumor cells into the lymph nodes and also the number of involved lymph nodes. These results highlight the importance of SALL4 and SOX2 gene expression in ESCC maintenance, progression and metastasis and emphasize the regulatory role of SALL4/SOX2 transcriptional network in the beginning and continuing of ESCC transformation, invasion and metastasis. Indeed, if we accept the existence of CSCs as a subgroup of tumor cells in ESCC, we have to consider such critical roles for these stem cell markers in establishment of the disease (Fig. 3d).

In conclusion, SALL4 functions in the ESCs (and probably CSCs) through a variety of protein–DNA and protein–protein interactions with crucial factors involved in maintenance of stemness state, self-renewal and pluripotency of the cell. It has not only a close cooperation with master regulators of stemness state including SOX2, OCT4 and NANOG, but also has a direct impact on major cell signaling pathways such as WNT/β-catenin in the cell, leading to cell survival and proliferation. To the best of our knowledge, the present study shows several interesting results for the first time in ESCC to date. We elucidated the co-expression pattern of two stemness regulatory transcription factors SALL4 and SOX2 in ESCC and found a significant correlation between the co-overexpression of these markers and invasion/metastasis of ESCC tumor cells. These data may confirm the existence and importance of CSCs in ESCC progression and development. The clinical consequences of SALL4–SOX2 association suggest a possible functional interaction between these factors in regulation of ESCC maintenance and aggressiveness and introduce them as potentially interesting therapeutic targets for planning an appropriate onco-therapy strategy to reverse a broad spectrum of functional aberrations that promote ESCC development.