Cytokeratin-17 expression is commonly observed in keratinocytic skin tumours and controls tissue homeostasis impacting HPV protein expression.

BACKGROUND: The structured expression of several keratins in the skin is associated with differentiation status of the epidermal layers, whereas others are upregulated only during wound healing, in skin disorders and in cancers. One of these stress keratins, K17, is correlated with poor prognosis in various cancer types and its loss has been shown to decelerate tumour growth. K17 expression can also be detected in cutaneous squamous cell carcinomas (SCCs), where UV-irradiation and infection with cutaneous human papillomaviruses (HPVs) are important co-factors. It was previously reported that K17 is upregulated in papillomavirus (PV)-induced benign skin lesions in mice and induces an immunological status that is beneficial for tumour growth. OBJECTIVES: In order to investigate whether K17 upregulation is induced by PVs, we analysed K17 levels in skin tumour specimens of different animal models and humans. METHODS: Various immunofluorescence stainings were performed to identify K17 expression as well as levels of E-Cadherin, vimentin and CD271. Tissues were further analysed by PCRs, qPCRs and ELISA to control for PV activity. K17knockdown cells were generated and effects on viral life cycle were investigated by infection assays, qPCR and Western blotting. RESULTS: We could show that K17 is commonly expressed in skin tumours and that its presence is not directly linked to viral oncoprotein expression. Rather, K17 expression seems to be a marker of epithelial differentiation and its absence in tumour tissue is associated with an epithelial-to-mesenchymal transition. We further showed that the absence of K17 in skin tumours increases markers of cancer stem-like cells and negatively affects viral protein synthesis. CONCLUSIONS: Collectively, our data indicate that K17 expression is a common feature in skin tumourigenesis. While it is not primarily targeted by PV oncoproteins, our in vivo and in vitro data suggest that it is an important regulator of epithelial differentiation and thus may play a role in controlling viral protein synthesis.

Viruses and phospholipids: Friends and foes during infection.

Viruses have evolved complex and dynamic interactions with their host cells to enable viral replication. In recent years, insights have been gained into the increasingly important role of the host cell lipidome in the life cycle of several viruses. In particular, viruses target phospholipid signaling, synthesis, and metabolism to remodel their host cells into an optimal environment for their replication cycle. Conversely, phospholipids and their associated regulatory enzymes can interfere with viral infection or replication. This review highlights examples of different viruses that illustrate the importance of these diverse virus-phospholipid interactions in different cellular compartments, particularly the role of nuclear phospholipids and their association with human papillomavirus (HPV)-mediated cancer development.

MAML1-induced HPV E6 oncoprotein stability is required for cellular proliferation and migration of cervical tumor-derived cells.

While a small proportion of high-risk (HR) alpha (α) human papillomaviruses (HPVs) is associated with numerous human malignancies, of which cervical cancer is the most prevalent, beta (ß) HPVs predominantly act as co-factors in skin carcinogenesis. A characteristic feature of both α- and ß-E6 oncoproteins is the presence of the LXXLL binding motif, which α-E6s utilize to form a complex with E6AP and which enables ß-E6s to interact with MAML1. Here we show that multiple α-E6 oncoproteins bind to MAML1 via the LXXLL binding motif and that this results in increased protein stability. Moreover, ß-E6 oncoprotein stability is also dependent on the interaction with MAML1. Additionally, in the absence of MAML1, endogenous HPV-8 E6 and HPV-18 E6 are rapidly degraded at the proteasome. Ablation of both E6AP and MAML1 leads to an even more profound downregulation of α-E6 protein expression, whereas this is not observed with ß-E6. This highly suggests that there is one cellular pool for most of ß-E6 that interacts solely with MAML1, whereas there are two cellular pools of HR α-E6, one forming a complex with MAML1 and the other interacting with E6AP. Furthermore, MAML1 induces HPV-8 E6 shuttling from the nucleus to the cytosolic fraction, while MAML1 interaction with HR E6 induces a drastic nuclear and membrane upregulation of E6. Interestingly, the HR α-E6/MAML1 complex does not affect targeting of some of the known HR E6 cellular substrates such as p53 and DLG1. However, MAML1 and E6AP joint co-expression with HR α-E6 leads to a significant increase in cellular proliferation, whereas silencing MAML1 decreases wound closure in HeLa cells. These results demonstrate that HR α-E6 interaction with MAML1 results in a stable form of E6, which likely modulates MAML1's normal cellular activities, one consequence of which being an increased proliferative capacity of HPV-transformed cancer cells. Thus, this study shows a novel function of the α-E6 oncoprotein and how it's activity might affect HPV-induced pathogenesis.

No Evidence for Role of Cutavirus in Malignant Melanoma.

Cutavirus was previously found in cutaneous melanoma. We detected cutavirus DNA in only 2/185 melanoma biopsies and in 0/52 melanoma metastases from patients in Germany. Viral DNA was localized in the upper epidermal layers. Swab specimens from healthy skin were cutavirus positive for 3.8% (9/237) of immunocompetent and 17.1% (35/205) of HIV-positive men.

Human papillomavirus type 8 oncoproteins E6 and E7 cooperate in downregulation of the cellular checkpoint kinase-1.

Human papillomavirus 8 (HPV8) is associated with the development of squamous cell carcinoma (SCC) of the skin. HPV-infected keratinocytes are able to override normal checkpoint control mechanisms and sustain cell cycle activity, allowing for synthesis of cellular proteins necessary for viral genome amplification. To study how HPV8 may disrupt cell cycle control, we analyzed the impact of HPV8 early gene expression on one of the key regulators of cell cycle and DNA damage response, checkpoint kinase-1 (CHK1). We found that expression of E1, E1̂E4, E2, E6 or E7 individually did not affect CHK1; however, keratinocytes expressing the complete early genome region (CER) of HPV8 showed a profound loss of CHK1 protein levels, that proved to be mediated by E6E7 co-expression. Neither CHK1 promoter regulation nor the ubiquitin-proteasome pathway are involved in HPV8-mediated CHK1 repression. However, CHK1 protein repression in organotypic skin cultures was paralleled by downregulation of the autophagy marker LC3B. Treatment of HPV8-CER expressing cells with the autophagy inhibitor Bafilomycin A1 rescued CHK1 expression and led to LC3B accumulation. Taken together, our data implicate that CHK1 autophagic degradation is enhanced by HPV8, which may contribute to the oncogenic potential of the virus.

BetaHPV E6 and E7 colocalize with NuMa in dividing keratinocytes.

Human papillomaviruses (HPVs) of genus betapapillomavirus (betaHPV) are implicated in skin carcinogenesis, but their exact role in keratinocyte transformation is poorly understood. We show an interaction of HPV5 and HPV8 oncoproteins E6 and E7 with the nuclear mitotic apparatus protein 1 (NuMA). Binding of E6 or E7 to NuMA induces little aneuploidy, cell cycle alterations, or aberrant centrosomes. Intracellular localization of NuMA is not altered by E6 and E7 expression in 2D cultures. However, the localization profile is predominantly cytoplasmic in 3D organotypic skin models. Both viral proteins colocalize with NuMA in interphase cells, while only E7 colocalizes with NuMA in mitotic cells. Intriguingly, a small subset of cells shows E7 at only one spindle pole, whereas NuMA is present at both poles. This dissimilar distribution of E7 at the spindle poles may alter cell differentiation, which may in turn be relevant for betaHPV-induced skin carcinogenesis.

HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer.

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV+ OPSCC metastasize faster than HPV- tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV+ OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44high /EpCAMhigh ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44high /EpCAMlow ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV+ OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44high /EpCAMlow CSCs were validated by RT-qPCR and in situ hybridization on HPV16+ OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16+ OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV+ and HPV- metastasis. As inhibition of this miRNA led to a markedly reduction of CD44high /EpCAMlow cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

The levels of epithelial anchor proteins ß-catenin and zona occludens-1 are altered by E7 of human papillomaviruses 5 and 8.

Infection with viruses of the genus Betapapillomavirus, ß-human papillomaviruses (ß-HPV), is implicated in the development of non-melanoma skin cancer. This was first evidenced for HPV5 and HPV8 in patients with the skin disease epidermodysplasia verruciformis (EV). The relocalization of the junctional bridging proteins ß-catenin and zona occludens-1 (ZO-1) from the adherens and tight junctions are common processes of the epithelial-mesenchymal transition (EMT) associated with tumour invasion. Here, we report that ß-catenin and ZO-1 are strongly upregulated by the E7 oncoproteins of HPV5 and HPV8 in keratinocytes grown in organotypic skin cultures. Although the membrane-tethered form of ß-catenin was elevated, no signs of ß-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of ß-catenin and ZO-1 could also be confirmed in the skin of HPV8 transgenic mice as well as in cutaneous squamous cell carcinomas of EV patients. These data provide the first evidence that ß-catenin and ZO-1 are direct targets of E7 of the oncogenic ß-HPV types 5 and 8. The ability to deregulate these epithelial junction proteins may contribute to the oncogenic potential of these viruses in human skin.

Human papillomavirus mediated inhibition of DNA damage sensing and repair drives skin carcinogenesis.

BACKGROUND: The failure to mount an effective DNA damage response to repair UV induced cyclobutane pyrimidine dimers (CPDs) results in an increased propensity to develop cutaneous squamous cell carcinoma (cSCC). High-risk patient groups, such as organ transplant recipients (OTRs) frequently exhibit field cancerization at UV exposed body sites from which multiple human papillomavirus (HPV)-associated cSCCs develop rapidly, leading to profound morbidity and increased mortality. In vitro molecular evidence indicates that HPV of genus beta-papillomavirus (ß-PV) play an important role in accelerating the early stages of skin tumorigenesis. METHODS: We investigated the effects of UV induced DNA damage in murine models of ß-PV E6 oncoprotein driven skin tumorigenesis by crossing K14-HPV8-E6wt mice (developing skin tumors after UV treatment) with K14-CPD-photolyase animals and by generating the K14-HPV8-E6-K136N mutant mouse strain. Thymine dimers (marker for CPDs) and γH2AX (a marker for DNA double strand breaks) levels were determined in the murine skin and organotypic skin cultures of E6 expressing primary human keratinocytes after UV-irradiation by immunohistochemistry and in cell lines by In Cell Western blotting. Phosphorylation of ATR/Chk1 and ATM were assessed in cell lines and organotypic skin cultures by Western blots and immunohistochemistry. RESULTS: Skin tumor development after UV-irradiation in K14-HPV8-E6wt mice could completely be blocked through expression of CPD-photolyase. Through quantification of thymine dimers after UV irradiation in cells expressing E6 proteins with point mutations at conserved residues we identified a critical lysine in the C-terminal part of the protein for prevention of DNA damage repair and p300 binding. Whereas all K14-HPV8-E6wt animals develop skin tumors after UV expression of the HPV8-E6-K136N mutant significantly blocked skin tumor development after UV treatment. The persistence of CPDs in hyperproliferative epidermis K14-HPV8-E6wt skin resulted in the accumulation of γH2AX foci. DNA damage sensing was impaired in E6 positive cells grown as monolayer culture and in organotypic cultures, due to lack of phosphorylation of ATM, ATR and Chk1. CONCLUSION: We showed that cells expressing E6 fail to sense and mount an effective response to repair UV-induced DNA lesions and demonstrated a physiological relevance of E6-mediated inhibition of DNA damage repair for tumor initiation. These are the first mechanistical in vivo data on the tumorigenicity of HPV8 and demonstrate that the impairment of DNA damage repair pathways by the viral E6 protein is a critical factor in HPV-driven skin carcinogenesis.

iASPP/p63 autoregulatory feedback loop is required for the homeostasis of stratified epithelia.

iASPP, an inhibitory member of the ASPP (apoptosis stimulating protein of p53) family, is an evolutionarily conserved inhibitor of p53 which is frequently upregulated in human cancers. However, little is known about the role of iASPP under physiological conditions. Here, we report that iASPP is a critical regulator of epithelial development. We demonstrate a novel autoregulatory feedback loop which controls crucial physiological activities by linking iASPP to p63, via two previously unreported microRNAs, miR-574-3p and miR-720. By investigating its function in stratified epithelia, we show that iASPP participates in the p63-mediated epithelial integrity program by regulating the expression of genes essential for cell adhesion. Silencing of iASPP in keratinocytes by RNA interference promotes and accelerates a differentiation pathway, which also affects and slowdown cellular proliferation. Taken together, these data reveal iASPP as a key regulator of epithelial homeostasis.

Expression of betapapillomavirus oncogenes increases the number of keratinocytes with stem cell-like properties.

Human papillomaviruses (HPV) of genus Betapapillomavirus (betaPV) are associated with nonmelanoma skin cancer development in epidermodysplasia verruciformis (EV) and immunosuppressed patients. Epidemiological and molecular studies suggest a carcinogenic activity of betaPV during early stages of cancer development. Since viral oncoproteins delay and perturb keratinocyte differentiation, they may have the capacity to either retain or confer a "stem cell-like" state on oncogene-expressing cells. The aim of this study was to determine (i) whether betaPV alters the expression of cell surface markers, such as CD44 and epithelial cell adhesion molecule (EpCAM), that have been associated with epithelial stemness, and (ii) whether this confers functional stem cell-like properties to human cutaneous keratinocytes. Fluorescence-activated cell sorter (FACS) analysis revealed an increase in the number of cells with high CD44 and EpCAM expression in keratinocyte cultures expressing HPV type 8 (HPV8) oncogenes E2, E6, and E7. Particularly through E7 expression, a distinct increase in clonogenicity and in the formation and size of tumor spheres was observed, accompanied by reduction of the epithelial differentiation marker Calgranulin B. These stem cell-like properties could be attributed to the pool of CD44(high) EpCAM(high) cells, which was increased within the E7 cultures of HPV5, -8, and -20. Enhanced EpCAM levels were present in organotypic skin cultures of primary keratinocytes expressing E7 of the oncogenic HPV types HPV5, -8, and -16 and in clinical samples from EV patients. In conclusion, our data show that betaPV may increase the number of stem cell-like cells present during early carcinogenesis and thus enable the persistence and accumulation of DNA damage necessary to generate malignant stem cells.

Tumor prevention in HPV8 transgenic mice by HPV8-E6 DNA vaccination.

The genus beta human papillomavirus 8 (HPV8) is involved in the development of cutaneous squamous cell carcinomas (SCCs) in individuals with epidermodysplasia verruciformis. Immunosuppressed transplant recipients are prone to harbor particularly high betapapillomavirus DNA loads, which may contribute to their highly increased risk of SCC. Tumor induction in HPV8 transgenic mice correlates with increased expression of viral oncogenes E6 and E2. In an attempt to prevent skin tumor development, we evaluated an HPV8-E6-DNA vaccine, which was able to stimulate a detectable HPV8-E6-specific cell-mediated immune response in 8/15 immunized mice. When skin of HPV8 transgenic mice was grafted onto non-transgenic littermates, the grafted HPV8 transgenic tissue was not rejected and papillomas started to grow within 14 days all over the transplant of 9/9 non-vaccinated and 7/15 not successfully vaccinated mice. In contrast, no papillomas developed in 6/8 successfully vaccinated mice. In the other two of these eight mice, a large ulcerative lesion developed within the initial papilloma growth or papilloma development was highly delayed. As the vaccine completely or partially prevented papilloma development without rejecting the transplanted HPV8 positive skin, the immune system appears to attack only keratinocytes with increased levels of E6 protein, which would give rise to papillomas.

HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses.

Despite epidermal turnover, the skin is host to a complex array of microbes including viruses, such as the human papillomavirus (HPV), which must infect and manipulate skin keratinocyte stem cells (KSC) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induces ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses (AK). Together these results define the "hit and run" mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lacks melanosome protection and is thus susceptible to sun-light-induced malignant transformation.

Human papillomavirus type 8 E6 oncoprotein inhibits transcription of the PDZ protein syntenin-2.

The E6 proteins from high-risk alpha human papillomavirus (HPV) types (e.g., HPV16) are characterized by the presence of a PDZ-binding motif through which they interact with a number of cellular PDZ domain-containing substrates and cooperate in their degradation. The ability of these E6 proteins to bind to PDZ domain proteins correlates with the oncogenic potential of the virus. The E6 proteins of oncogenic HPV from the genus Betapapillomavirus (betaPV, e.g., HPV8) do not encode a PDZ-binding motif. We found that the PDZ domain protein syntenin-2 is transcriptionally downregulated in primary human epidermal keratinocytes (PHEK) by HPV8 E6. The mRNA levels of the known HPV16 E6 PDZ protein targets Dlg, Scribble, Magi-1, Magi-3, PSD95, and Mupp1 were not changed by HPV8 E6. Decreased protein levels of syntenin-2 were observed in cell extracts from PHEK expressing HPV5, -8, -16, -20, and -38 E6 but not in HPV1 and -4 E6-positive keratinocytes. Surprisingly, HPV16 E6 also repressed transcription of syntenin-2 but with a much lower efficiency than HPV8 E6. In healthy human skin, syntenin-2 expression is localized in suprabasal epidermal layers. In organotypic skin cultures, the differentiation-dependent expression of syntenin-2 was absent in HPV8 E6- and E6E7-expressing cells. In basal cell carcinomas of the skin, syntenin-2 was not detectable, whereas in squamous cell carcinomas, expression was located in differentiated areas. Short hairpin RNA-mediated knockdown of syntenin-2 led to an inhibition of differentiation and an increase in the proliferation capacity in PHEK. These results identified syntenin-2 as the first PDZ domain protein controlled by HPV8 and HPV16 at the mRNA level.

Poly(I:C) Treatment Prevents Skin Tumor Formation in the Preclinical HPV8 Transgenic Mouse Model.

Actinic keratoses and cutaneous squamous cell carcinomas are associated with infections with human papillomavirus of genus beta (betaHPV) in immunosuppressed patients. To date, targeted therapy against betaHPV-associated skin cancer does not exist because of the large number of betaHPV without defined high-risk types. In this study, we hypothesized that the activation of innate antiviral immunity in the skin, asymptomatically infected with betaHPV, induces an antitumor response by in situ autovaccination and prevents the formation of betaHPV-associated skin cancer. To test this, we used the preclinical keratin-14-HPV8 transgenic mouse model, which develops skin tumors after mechanical wounding. Remarkably, treatment with the antiviral immune response activating polyinosinic-polycytidylic acid (poly[I:C]) completely prevented cutaneous tumor growth. The induction of the IFN-induced genes Cxcl10 and Ifit1 by poly(I:C) depended on MDA5 activation. Increased numbers of total and activated CD4 and CD8 T cells were detected in poly(I:C)-treated skin. T cells were found in the skin of poly(I:C)-treated mice but not in the skin tumors of untreated mice. T-cell depletion showed a predominant role of CD4 T cells in poly(I:C)-mediated tumor prevention. Our findings identify the MDA5 ligand poly(I:C) as a promising candidate for in situ autovaccination approaches, which might serve as a treatment strategy against betaHPV-related skin diseases.

HPV8 Reverses the Transcriptional Output in Lrig1 Positive Cells to Drive Skin Tumorigenesis.

K14-HPV8-CER transgenic mice express the complete early genome region of human papillomavirus type 8 (HPV8) and develop skin tumours attributed to the expansion of the Lrig1+ stem cell population. The correlation between HPV8-induced changes in transcriptional output in the stem cell compartment remains poorly understood. To further understand the oncogenic pathways underlying skin tumour formation we examined the gene expression network in skin tumours of K14-HPV8-CER mice and compared the differentially expressed genes (DEG) with those of the Lrig1-EGFP-ires-CreERT2 mice. Here, we report 397 DEGs in skin tumours of K14-HPV8-CER mice, of which 181 genes were up- and 216 were down-regulated. Gene ontology and KEGG pathway enrichment analyses suggest that the 397 DEGs are acting in signalling pathways known to be involved in skin homeostasis. Interestingly, we found that HPV8 early gene expression subverts the expression pattern of 23 cellular genes known to be expressed in Lrig1+ keratinocytes. Furthermore, we identified putative upstream regulating transcription factors as well as miRNAs in the control of these genes. These data provide strong evidence that HPV8 mediated transcriptional changes may contribute to skin tumorigenesis, offering new insights into the mechanism of HPV8 driven oncogenesis.

Human Beta Papillomavirus Type 8 E1 and E2 Proteins Suppress the Activation of the RIG-I-Like Receptor MDA5.

Persistent infections of the skin with the human papillomavirus of genus beta (ß-HPV) in immunocompetent individuals are asymptomatic, but in immunosuppressed patients, ß-HPV infections exhibit much higher viral loads on the skin and are associated with an increased risk of skin cancer. Unlike with HPV16, a high-risk α-HPV, the impact of ß-HPV early genes on the innate immune sensing of viral nucleic acids has not been studied. Here, we used primary skin keratinocytes and U2OS cells expressing HPV8 or distinct HPV8 early genes and well-defined ligands of the nucleic-acid-sensing receptors RIG-I, MDA5, TLR3, and STING to analyze a potential functional interaction. We found that primary skin keratinocytes and U2OS cells expressed RIG-I, MDA5, TLR3, and STING, but not TLR7, TLR8, or TLR9. While HPV16-E6 downregulated the expression of RIG-I, MDA5, TLR3, and STING and, in conjunction with HPV16-E7, effectively suppressed type I IFN in response to MDA5 activation, the presence of HPV8 early genes showed little effect on the expression of these immune receptors, except for HPV8-E2, which was associated with an elevated expression of TLR3. Nevertheless, whole HPV8 genome expression, as well as the selective expression of HPV8-E1 or HPV8-E2, was found to suppress MDA5-induced type I IFN and the proinflammatory cytokine IL-6. Furthermore, RNA isolated from HPV8-E2 expressing primary human keratinocytes, but not control cells, stimulated a type I IFN response in peripheral blood mononuclear cells, indicating that the expression of HPV8-E2 in keratinocytes leads to the formation of stimulatory RNA ligands that require the active suppression of immune recognition. These results identify HPV8-E1 and HPV8-E2 as viral proteins that are responsible for the immune escape of ß-HPV from the innate recognition of viral nucleic acids, a mechanism that may be necessary for establishing persistent ß-HPV infections.

Impact of Human Papillomavirus on Wnt/Beta-Catenin Signaling in Morphological Inconspicuous Cervicovaginal Cells.

INTRODUCTION: The aim of this study was to identify early changes in the Wnt/beta-catenin signaling pathway in high-risk human papillomavirus (HPV) infected cervicovaginal cells and to correlate these changes with cell proliferation, apoptosis, and autophagic processes. METHODS: We evaluated 91 cervicovaginal smears of women with (n = 41) and without (n = 50) HPV-DNA. Smears were stained against beta-catenin, c-myc, secreted frizzled-related protein 4 (sFRP4), cleaved caspase-3, and the autophagy markers Beclin-1 and light chain 3B. In addition, sFRP-1, -2, -3, -4, -5 mRNA levels were determined by quantitative reverse transcription-PCR in primary keratinocytes and FaDu cells expressing HPV16-E6, -E7, or -E6E7. RESULTS: Our data indicated that the Wnt/beta-catenin signaling is activated in HPV (+) cervicovaginal cells that can already be detected in cells with no obvious changes in cellular morphology (HPV [+]/cyto [-]). These cells also had significantly higher sFRP4 levels when compared to HPV-negative samples. In primary keratinocytes, sFRP4 was found to be absent and sFRP1 and sFRP2 to be repressed in the presence of HPV16-E6 and E7. Interestingly, sFRP4 is expressed in FaDu cells and can be upregulated in the presence of E6E7. Curiously, SFRP4 expression correlated with an increase in the level of autophagic markers in HPV (+)/cyto (-) smears. CONCLUSION: In conclusion, the activation of the Wnt/beta-catenin signaling pathway and upregulation of sFRP4, paralleled by an activation of the autophagic pathway may represent predisposing cellular factors early after HPV infection which need to be further determined in larger study.