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

Bovine papillomavirus (BPV) is a worldwide oncogenic virus, present in at least 60% of Brazilian cattle herd ^{1, 2}. The BPV is the etiological agent of bovine papillomatosis (BP), infectious and neoplastic disease, characterized by the presence of multiple papillomas (warts) that can regress spontaneously or to persist and progress to urinary bladder or upper alimentary tract carcinoma when in association with environmental cofactors, such as the quercetin and ptaquilosides found in the break fern Pteridium ssp.^{2, 3, 4, 5}.

Although recognized that the BPV promotes cytogenetic aberrations ^{1, 6}, which are related to the viral clastogenic potential ^{7, 8, 9}, the viral action following cancer initiation remains few explored ¹⁰. 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). The EMT is a complex pathological process verified during metastasis, characterized by reversible metabolic, genetic and morphological reprograming ^{10, 20, 22, 23}, which can lead to cancer stem-cell (CSC) formation ^{23, 24, 25, 26}.

Based on these data, this study aimed to evaluate the expression of cytokeratin 10 (CK10), STAT3 Y705, Oct-3/4 and vimentin in paraffin-embed tissue samples from BPV-infected cutaneous papilloma (papilloma 01), fibropapilloma (papilloma 02 and 03) and esophageal carcinoma and BPV-free normal skin (negative control) that originated the cell lines used in previous studies to validate the results previous verified in cell culture.

In last years, studies involving the EMT and CSC formation have been awakened attention. This because the EMT and CSC are intrinsically associated, since the biochemical and genetic reprograming that lead to the mesenchymal phenotype acquisition can also promote the cell dedifferentiation, resulting in the stem-cell acquisition ^{24, 25, 29}. However, while the cell reprograming that lead to EMT is dependent on reversible epigenetic changes, the genetic alterations verified in this process can be irreversible, contributing with the CSCs development ³⁰.

The CSCs represent one of the major challenges to be overcome in oncology field, once these cells are refractory to chemotherapeutics ³¹ and are related to the metastasis formation ³². Considering the Darwinian dynamic of cancer, the acquisition of both mesenchymal and stem-cell phenotype confers competitive advantages for CSCs, which exhibit a most efficient repair system, making these cells less sensitive to antineoplastic drugs and/or radiotherapy ^{32, 33}. These data are alarming, since the CSCs can comprise until 25% of the cancer cell content ³⁴. Moreover, due to the capability to divide asymmetrically, these cells can self-renewal, maintain these cell subpopulation into tumor microenvironment ³⁵.

The genomic instability is recognized as drives the carcinogenesis by induce the cancer initiation ^{36, 37}. However, current studies have been also shown that genomic instability is related to CSCs formation ^{38, 39}. In this sense, the BPV is an oncogenic virus known as induce DNA damages in host chromatin, leading to genomic instability ^{1, 7, 40}. Despite this, the BPV action following cancer initiation remains unresolved ¹⁰. The lack of studies about the viral role in metastasis can be attributed to the replication viral paradigm, which is dependent on epithelium differentiation ⁴¹. For this reason, little attention has been given to cell lines derived from BPV-infected neoplasms. Moreover, studies involving paraffin embed tissues focus on the immunodetection of viral proteins or oncoproteins and their interaction with host proteins ^{9, 42, 43}.

In this context, in 2008 we established cell lines derived from BPV-infected cutaneous papilloma, fibropapilloma and esophageal carcinoma ¹¹, bringing new opportunities to interrogate the BPV action in cancer progression and metastasis. Using these cell lines, we already verified that the viral infection persistence can lead promote the glycolytic metabolism 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.

First, we analyzed the tissue architecture through histopathological analysis. Results of this analysis showed the preservation of epithelial and dermal histology of normal skin (Figure 1). Considering the results of PCR and BPV E5 immunodetection (Figure 2 and Figure 3, respectively), we confirmed the absence of BPV infection in this sample, allowing its use as negative control. However, the samples of papilloma 01-03 showed a notorious hyperplasia of suprabasal layer (acanthosis) (Figure 1). Acanthosis is consequence of proliferative action of E5, E6 and E7 oncoproteins, that combined stimulates the S phase entry, making available the DNA polymerases required for BPV replication. Considering that BPV is able to promote the oxidative stress ^{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.

The samples of papilloma 01-03 also showed a hyperkeratosis (Figure 1). Hyperkeratosis confers a physical protection for virions, at the same time that avoid an immune response, since the icosahedral morphology of BPV capsid is immune-reactive. ². The papilloma 03 also showed a high quantity of kerato-hyaline granules on granular layer (Figure 1). The presence of these granules was first identified in 1869 by Auffhame and, later, described in 1873 by 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}. Thus, the koilocytes verified in papillomas 01-03 samples indicate the BPV infection in these tissues, which was proved by the PCR results and the BPV E5 oncoprotein immunodetection (Figure 2 and Figure 3, respectively). Koilocytosis represents a cytopathic effect of viral infection ⁵⁰, being observed in papillomas of different species, including humans ^{47, 48, 51, 52}. Although the pathways involved in perinuclear vacuolization are unclear, it is believed that it could contribute to cell fragility, facilitating the virion release to environment ⁵³.

However, the samples of papilloma 02 and 03 showed a fibro-elastic reactive stroma, characterized by the intense fibroblastic proliferation (Figure 1). For this reason, these samples were classified as fibropapilloma. Fibropapilloma is histologically similar to equine sarcoid, fibroblastic benign neoplasm, locally invasive but not metastatic, that until 1963 was known as equine fibropapilloma ^{54, 55, 56, 57}. Fibropapilloma are associated to BPV-1, 2 and 13 (Deltapapillomavirus) and BPV-5 (Epsilonpapillomavirus) infection, being also described in zebra (Equus burchelli), horse, buffalo llama, camel, alpaca, dumb and cats infected by BPV, as well as by ovine infected by OvPV-1 and 2 ^{56, 58, 59, 60, 61, 62, 63}.

Esophageal carcinoma showed a tissue disorganization (Figure 1), which can be attributed to cell proliferation. Histopathological results also showed a reactive dermis, whith epithelioid cells islands in dermaepidermla junction (Figure 1). These islands suggest an invasiviness and migratory phenotype confered by the EMT. Furtheremore, it was verified the presence of fibroblastoid cells into these islands (Figure 1), indicating the loss of apical-basal polarity, suggesting the acquisition of EMT phenotype. Similar results were described in the cell lines derived from this tissue ²¹.

Aiming to confirm the BPV infection, we performed the immunodetection of E5 oncoprotein. The E5 is a small hydrophobic transmembrane protein ^{64, 65}, recognized as the main transforming protein ⁶⁶. The E5 oncoprotein can bind and promote the PDGFβR dimerization ^{64, 67, 68}. This action lead to activation of different kinases (A-cdk2, MAPK, JNK, PI3K e c-Src), resulting in cell proliferation and cancer 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.

We verified the E5 oncoprotein expression on plasmatic membrane and cytoplasm of basal and suprabasal keratinocytes of papilloma 01, 02, 03 and esophageal carcinoma samples (Figure 2), but not in BPV-free normal skin (Figure 2). This data reinforces the viral infection, previous verified in cell lines derived from these tissues ¹².

Considering that during the EMT occur the repression of epithelial markers, we analyzed the expression levels of cytokeratin 10 (CK10). The CK10 is one the most analyzed cytokeratin in pathology, since it is recognized as a marker of cell differentiation, being expressed from suprabasal to corneal epithelium layer ^{72, 73, 74}. The immunodetection of CK10 has been commonly used in histopathological routine, once alterations in CK10 expression levels are observed in carcinomas ^{73, 75, 76, 77, 78}. These alterations are frequently associated with metastasis in human ^{75, 77} and bovines ⁷⁶, making the CK10 a prognostic marker since 1980 ^{75, 77, 79}.

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. For this reason, cytokeratins form heterodimers comprised by type I and II CKs, conferring mechanical stability to the cells ^{75, 79, 80}. Thus, the results showed a notorious reduction in CK10 expression levels in fibropapilloma and esophageal carcinoma in relation to BPV-free normal skin and cutaneous papilloma (Figure 3). This result is very interest, once the fibropapillomas showed an acanthosis similar to cutaneous papilloma (Figure 1), should be expected similar levels of CK10 expression in both cutaneous papilloma and fibropapilloma. Thus, considering that the cancer emerges from a pre-neoplastic lesion, these results suggest that fibropapilloma can be discussed as a pre-malignant lesion. These data are in accordance to the previous results obtained from cell lines derived from the tissues samples used in this study, that 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 observed an expressive immunodetection of STAT3 Y705 in the dermo-epidermal junction of cutaneous papilloma, fibropapilloma and esophageal carcinoma (Figure 4). Curiously, the cells expression STAT3 Y705 into the epidermis exhibited a fibroblastoid morphology, showing the loss of apical-basal polarity, while cells expressing this factor into the dermis showed a keratinocyte-like morphology (Figure 4). These results are in consonance with our previous data, verified using 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.

Considering these results, that combined suggest a cell dedifferentiation along the natural history of cancer development, we analyzed the expression levels of Oct-3/4 (POU5F1), which is recognized as marker of CSC ⁹⁰. The results of this analysis showed an expressive nuclear immunodetection of Oct-3/4 in dermo-epidermal junction of cutaneous papilloma and fibropapilloma (Figure 5), exhibiting a pattern similar to those verified in immunodetection of STAT3 (Figure 4). However, it was not observed the expression of Oct-3/4 in the esophageal carcinoma (Figure 5). Although controversial, this result can be attributed to the high mitogenic activity verified in the esophageal carcinoma, verified by the presence of mitotic cells (Figure 5). Moreover, this data reinforces that the main alterations verified in the tumor microenvironment occur from a pre-malignant stage.

The CSC phenotype acquisition confers resistance to apoptosis in cells genetically unstable. In this context, the BPV E6-mediated p53 downregulation confers an additional anti-apoptotic action, at the same time that induce DNA damages ⁹¹. These combined action contribute with both cancer initiation, increasing the entropic status ³³ and, metastasis, conferring anoikis resistance, which is recognized as a EMT hallmark ⁹². On the one hand the CSC phenotype acquisition is mandatory to distant metastasis formation, on the other hand, it represents a challenge for therapeutics.

Added to these results, we verified the immunodetection of vimentin (VIM) in dermis in all tissues analyzed (Figure 6). As expected the samples of papilloma 02 and 03 (fibropapilloma) showed an expressive expression of VIM into the dermis, confirming the BPV fibrotropism described in the histopathological analysis (Figure 1). However, it was also verified the VIM expression in keratinocytes present in both dermo-epidermal junction and subrabasal epithelium layer (Figure 6). The VIM overexpression has been reported in HPV-related head and neck squamous cell ^{93, 94}, cervical ²² and penile carcinomas ⁹⁵.

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. Changes in the extracellular matrix (ECM) are also mandatory to allow the transformed cell migration. For this reason, we also analyzed the collagen composition of ECM using the Picrosirius red staining, method developed by Constantine and Mowry in 1968 ¹⁰¹. This method allows to identify collagen fibers of type I and II ²⁷, which are crucial for the tissue integrity maintenance ¹⁰². Under a wave-lenght of 540 nm, collagen type I fibers are displayed in red-orange color, while fibers of type III, yellow-green ²⁷.

Results of this analysis showed a significant reduction in collagen type III fibers and a consecutive increase in type I fibers in samples from cutaneous papilloma, fibropapillomas and esophageal carcinoma in relation to the BPV-free normal skin (Figure 7). These data reinforces that the BPV can promote the ECM remodeling, leading to EMT. This because the collagen type I fibers can activate focal adhesion kinase (FAK), resulting in the E-cadherin dissociation, that is recognized as a EMT hallmark ^{103, 104}. This process results in the nuclear translocation of β-catenin, promoting the E-cadherin downregulation ¹⁰⁵.

In summary, these combined 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. 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).

The authors thank the collaboration with Prof. Dr. Franco Roperto (University of Naples Federico II) and the Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, Grant number 2014/20617-5) by the financial support.

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