Several lines of evidence show that EMT has a central role in invasion and metastasis of different types of tumours facilitating conversion of polarized, epithelial phenotype to motile fibroblastoid or mesenchymal phenotype [6
]. Recently EMT markers have also been associated with early steps of tumourigenesis [8
]. The aim of this study was to elucidate the role of transcription factor Snail1, an inducer of EMT, in the development of epithelial ovarian tumours.
According to our results nuclear expression of Snail1 protein seems to be a feature of tumour progression in epithelial ovarian tumourigenesis. Increased Snail1 expression was associated with increasing tumour malignancy both in epithelial compartment (p = 0.006) and in stromal compartment (p = 0.007). Our result is in concordance with previous studies indicating Snail1 as a regulator of EMT as well as literature describing it as an inducer of invasive phenotype [10
]. However, Snail1 protein may have additional roles in different types of tumours.
Nuclei of all benign tumours were negative for Snail1. In line with our results one report presented negative Snail1 expression in mouse intestinal adenomas, and another in human hyperplastic oral mucosa [19
]. However, dysplastic oral mucosa has been reported to show single distributed Snail1 positive cells [19
]. We found similarly focally positive nuclear Snail1 protein staining in borderline ovarian tumours. Snail1 expression in precursor lesions may suggest a role for the protein in early ovarian tumourigenesis before invasion or metastasis. Its expression in borderline tumours might be related to other known functions of Snail1. Indeed, members of Snail1 family can also act as more general regulators of cell adhesion and movement in EMT-related or EMT-independent manner [26
]. In cancer Snail1 has been described to act primarily as a survival factor and an inducer of cell movement rather than as an inducer of EMT [27
]. Peinado et al. have proposed that Snail1 could be implicated in the initial migratory phenotype of primary tumours and considered it as an early marker of EMT, at least in ovarian tumours [10
In the vast majority of the Snail1 positive ovarian carcinomas the nuclear protein was stained focally at rather low levels in the tumour epithelium and stroma. Nuclear content of Snail1 in epithelium or stroma in our material was not related to clinicopathological features, like tumour grade or stage of disease, nor had it prognostic significance. Relatively small material and short follow-up time of the patients could effect on this. However, nuclear staining was detected in borderline tumours suggesting that Snail1 might be an early factor in ovarian tumour development. Nevertheless, supporting our findings rather similar results have been reported [16
]. In another ovarian cancer study using the material of 48 primary tumours nuclear Snail1 staining in epithelial cells in 38% of the primary tumours did not associate with any clinicopathological factors or overall survival [16
Stromal cells are essential in tumourigenesis [28
]. Presence of Snail1 positive stromal cells has previously been detected in various adeno and squamous cell carcinomas [14
]. In our study, similar staining of Snail1 protein was observed both in stromal fibroblast-like fusiform cells and epithelial cells of ovarian carcinomas. Thus, a tumour may create its own stroma originating from malignant epithelium to facilitate tumour growth [29
] and Snail1 could be a marker of these cells that have just undergone EMT [14
]. Indeed, in vitro
Snail1-transfected (squamous) carcinoma cells show complete EMT phenotype with fibroblastic characteristics [11
]. However, in the present study expression of Snail1 in stromal cells was also detected in borderline tumours that do not have histological evidence of stromal invasion indicating that Snail1 may have also other functions in stromal fibroblast-like fusiform cells [27
]. Snail1 has also been identified as a regulator of terminally differentiated mesenchymal cells [30
], and further, during tumourigenesis, Snail1 expression in stromal cells may indicate interactions of fibroblasts with dedifferentiated tumour cells [19
]. Snail1-induced EMT has been proposed also to accelerate cancer metastasis by enhancing invasion and by inducting immunosuppression [31
]. Altogether, Snail1 expression in the stroma of borderline tumours may indicate importance of the microenvironment already in premalignant tumours and supports the role of tissue remodelling effect of Snail1.
In tumours, endothelial to mesenchymal transition (EndMT) is an important source of cancer-associated fibroblasts [32
]. Some of the endothelial cell nuclei in this ovarian carcinoma study were positive for Snail1. Snail1 staining in a subset of endothelial cells has been reported in adeno and squamous cell carcinomas [15
]. However, we found positive endothelial cells also in benign and borderline tumours as well as in normal ovaries. A previous report has demonstrated that angiogenic vessels can undergo EndMT [32
]. Kokudo et al. showed that mouse endothelial cells can differentiate into mural cells, such as pericytes and/or smooth muscle cells, and that Snail1 is associated with the process [33
]. Also in normal human ovaries new blood vessel formation and subsequent regression occurs in each hormonal cycle [34
Besides nuclear protein content we have also analysed cytoplasmic staining of Snail1 protein in epithelial cells. Opposite to nuclear immunoreactivity, we observed decreased cytoplasmic expression of Snail1 in epithelium that was significantly associated with increasing tumour malignancy (p < 0.001). Transcription factor Snail1 activity is controlled by subcellular location and cytoplasmic form of the protein is not considered active [10
]. Keeping this in mind, cytoplasmic staining may point to post-transcriptional regulation of the protein in epithelial ovarian carcinomas [10
] and in pre-malignant tumours, as shown in the present material.