First Evidences of Epithelial-Mesenchymal Transition and Cancer Stem-Cell Phenotype Acquisition in Dermo-Epidermal Junction of BPV-Infected Neoplasms

Introduction: Bovine papillomavirus (BPV) is the etiological agent of bovine papillomatosis, infectious and neoplastic disease, characterized by the presence of multiple papillomas that can regress spontaneously or to persist and progress to malignancies when in association with environmental cofactors. Although recognized that the BPV can induce DNA damages, the viral role following cancer initiation remains unresolved. Based on this, we stablished cell lines derived from cutaneous papilloma, fibropapilloma and esophageal carcinoma to study the BPV action on epithelial-mesenchymal transition (EMT). Our results showed strong evidences that the virus action can contribute to EMT and, therefore, metastasis. Aim: In this study, we analyzed the expression levels of the EMT markers (cytokeratin 10, STAT3 Y705, Oct-3/4 and vimentin) in paraffin-embed samples, using the same tissues that originated the cell lines previous studied, aiming to validate the results observed using cell lines. Material and Methods: Expression levels of these markers was analyzed by immunohistochemistry and the collagen composision by Picrosirius red staining. Results: We verified an overexpression of these markers in fibroblastoid cells present into the epidermis and ketarinocyte-like cells into the dermis present in dermo-epidermal junction. These data reinforce our previous results using cell cultures, validating both systems (cell culture and paraffin-embed tissues) as useful models to study the natural history of BPV-infected lesions. Conclusion: Altogether, the results from these systems indicate that the BPV promote the cancer progression and metastasis through the transdifferentiation of an epithelial to mesenchymal cells (EMT). DOI : 10.1302/issn.2576-6694.jbbs-17-1869 Corresponding Author : Rita de Cassia Stocco, Genetics Laboratory, Butantan Institute, 1500, Vital Brazil Avenue, São Paulo, Brazil, Zip code 055000-900, Phone/Fax: +55 11 2627-9701 e-mail: rita.stocco@butantan.gov.br

The lack of studies about the BPV on cancer progression and metastasis can be attributed to the lack of attention given to cell cultures derived from BPV-infected tissues, which are mandatory to interrogate the viral action following cancer initiation.
In this sense, since 2008, when we established with success different cell lines derived from BPV-infected benign and malignant neoplasms [11], we have been explored the potential of these cell lines as model to study the BPV-related oncogenic process.
Using these cell lines, we already showed that they exhibit cytogenetic alterations [3,12] similar to those verified in short-term lymphocyte cultures derived from BPV-infected animals [1,6,7]. Similar data were also described in primary cell cultures derived from BPV-infected papilloma and equine sarcoid [13]. These results suggest that cell lines derived from BPV-infected neoplasms are useful model to study the viral action on cancer initiation.
Currently we also described the BPV L1 capsid protein expression and the presence of virus-like particles in cytoplasmic vesicles of cells in sixth passage derived from cutaneous papilloma, fibropapilloma and esophageal carcinoma [14], suggesting that these cell lines is also an useful model to study the viral biology.
In addition, we verified that BPV can induce metabolic alterations in cell lines derived from fibropapilloma and esophageal carcinoma as a possible consequence of anti-oxidant action of BPV E6 oncoprotein [12]. These deregulations in energetic metabolism lead to reactive oxygen species (ROS) production, inducing the DNA oxidation. This action can result in DNA breaks (clastogenesis) and, to activate the nuclear transcription factor STAT3 through the phosphorylation of tyrosine residue 705 (Y705) [15]. When activate, the STAT3 Y705 promotes cell proliferation [16] by the activation of mitogen-activated protein kinase (MAPK) [17] and platelet-derived growth factor beta (PDGFβ) upregulation [18]. The STAT3 activation also induces the mesenchymal proteins expression, including vimentin and, regulates the cell differentiation [19,20].
Thus, these data can justify the overexpression of STAT3 Y705, Oct-3/4 and vimentin verified in cell lines derived from BPV-infected neoplasms [21]. Altogether, these alterations suggest that BPV infection persistence can lead to epithelial-mesenchymal transition (EMT).
Based on these data, this study aimed to

Histopathological Analysis
Histopathological      remodeling, contributing to cell migration and, therefore, which EMT.

Discussion
In last years, studies involving the EMT and CSC activation and increase the ROS production [12]. We also verified that BPV induces the activation and overexpression of STAT3 and Oct-3/4, leading to the loss of cell polarity, cell migration and formation of oncospheres [12,21]. Now, we investigate if these alterations are also present in the tissue samples that originated these cell lines.   [10,12], acanthosis can be also discussed as a consequence of ROS production, which, under determinate concentrations, can lead to cell proliferation as previously described in equine sarcoid infected by BPV-1 and 2 [44] and in bovine fibropapilloma [9]. Langerhans [45]. These granules are rich of cysteine, amino acid characterized by the presence of sulfhydryl group responsible for the disulfide bond, involved in keratinization process [45,46].
Considering the natural history of BPV replication cycle, the viral particles are release from the keratinocytes in degeneration present in suprabasal and granular layer. These cells exhibit a prominent halo and an acentric nucleus, being known as koilocytes [47,48].
The presence of these cells is considered for some authors as a pathognomonic marker of papillomavirus infection [47,49]  progression [64,69,70]. The E5 oncoprotein also promotes the loss of focal adhesion, leading to invasion [71]. Thus, the E5 is also related with EMT and, therefore, with CSC formation.
Cytokeratin is a class of evolutionary conserved structural proteins [80], encoded by different genes and classified in two types: type I (acid polypeptides with 40-56 kDa, including the CK9 to CK20) and type II (neutral and basic polypeptides with 53-68 kDa, including CK1 to CK8) [75]. The CK monomers are fastly degradated [75]. showed that fibropapillomas exhibit metabolic alterations [12] and an intermediate migration capability in relation to the BPV-free normal skin and esophageal carcinoma [21]. Altogether, these data reinforce our hypothesis that fibropapillomas are pre-malignant lesion.
Results also showed an increase in STAT3 Y705 expression levels in all BPV-infected lesions in relation to BPV-free normal skin ( figure 4). The STAT3 is a member of Signal Transducer and Activator of Transcription (STAT) family of nuclear transcriptional factor [81].
These factor regulate different target genes, leading to cell proliferation, differentiation, apoptosis, migration, angiogenesis and anoikis resistance [81]. The STAT3 nuclear factor is crucial for the maintenance of tissue integrity, regulating the epithelial-mesenchymal interaction [82,83]. However, the aberrant expression/activation of STAT3 is related to the malignant transformation and metastasis formation [16,81].
The STAT3 protein remains latent in the cytosol [18], where is activated by the Janus kinase (JAK) [84], which promotes the phosphorilation of (1) 727 serine residue present in the transactivation domain (S727) or (2) 705 tirosine residue presente in C-terminal domain (Y705) [83]. Current studies have been shown that the Y705 phosphorilation is the main pathway of STAT3 activation [83,85]. For this reason, we analyzed the levels of expression of STAT3 Y705. We the cell lines derived from these tissues [12].
The STAT3 activation also lead to the CSC formation [83], justifying the repression of CK10 expression observed in fibropapilloma and esophageal carcinoma ( figure 3). However, the activation of STAT3 is not restricted to cells infected by BPV. Studies have been described that other oncogenic viruses, such as the Epstein-Barr virus (EBV) [86], Rous sarcoma virus (RSV) [87], human T-leukemia virus (HTLV) [88], and the hepatites B (HBV) and C viruses (HCV) [89] can also promote the STAT3 phosphorilation. Altogether, these results suggest that the STAT3 activation is a carcinogenic pathway shared among oncogenic viruses.
For this reason, to explore drugs able to modulate this pathway can be considered a plausible alternative to avoid the cancer development or, control the cancer progression and consequent metastasis.
Vimentin is one of the most abundant mesenchymal protein expressed in mammals [96]. The VIM is evolutionary conserved [20], for this reason, the anti-vimentin clone V9 antibody has been used for the vimentin detection in bovines [97,98]. The protein confers mechanic-structural support for mesenchymal cells [99] and has multiple phosphorilation sites [20], which act as substrate for different kinases, including the Rho kinases [99,100]. The phosphorilation of these sites leads to VIM depolymerization, confering cell motility [99]. Due to these reasons, the overexpression of VIM is considered a canonical marker of EMT [20].
However, the alterations that lead to EMT are not restrict to cell genetic and epigenetic deregulations.