Martin Hufbauer

Spontaneous tumour development in human papillomavirus type 8 E6 transgenic mice and rapid induction by UV-light exposure and wounding

Cutaneous human papillomavirus type 8 (HPV8) is carcinogenic in patients with epidermodysplasia verruciformis. Transgenic mice with the complete early region (CER) of HPV8 spontaneously developed papillomas, dysplasia and squamous cell carcinomas of the skin. To characterize the role of individual early genes in carcinogenesis, the E6 and E6/E7 genes were expressed separately in transgenic mice. Nearly all HPV8-E6-positive mice spontaneously developed multifocal tumours, characterized by papillomatosis, hyperkeratosis and varying degrees of epidermal dysplasia. In 6 % of the cases, the tumours became malignant, comparable with HPV8-CER mice. Thus, in the murine epidermis, E6 is the major oncogene necessary and sufficient to induce spontaneous tumour development up to the level of squamous cell carcinoma. To evaluate the synergistic effects of UV light and wound healing, the skin of HPV8 mice was irradiated with UVA/UVB light or wounded with punch biopsies. These treatments induced papillomatosis in HPV8-CER and -E6 mice within 3 weeks. Irradiation with UVA alone did not induce papillomatosis and UVB alone had a weaker effect than UVA/UVB, indicating a synergistic role of UVA in UVB-induced papillomatosis. An HPV8 infection persisting over decades in interaction with sun burns and wound healing processes may be a relevant cause of skin cancer in humans.

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

ABSTRACT 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 CD44high EpCAMhigh 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.

Enhanced human papillomavirus type 8 oncogene expression levels are crucial for skin tumorigenesis in transgenic mice

Human papillomavirus 8 (HPV8) is involved in skin cancer development in epidermodysplasia verruciformis patients. Transgenic mice expressing HPV8 early genes (HPV8-CER) developed papillomas, dysplasias and squamous cell carcinomas. UVA/B-irradiation and mechanical wounding of HPV8-CER mouse skin led to prompt papilloma induction in about 3 weeks. The aim of this study was to analyze the kinetics and level of transgene expression in response to skin irritations. Transgene expression was already enhanced 1 to 2 days after UVA/B-irradiation or tape-stripping and maintained during papilloma development. The enhanced transgene expression could be assigned to UVB and not to UVA. Papilloma development was thus always paralleled by an increased transgene expression irrespective of the type of skin irritation. A knock-down of E6 mRNA by tattooing HPV8-E6-specific siRNA led to a delay and a lower incidence of papilloma development. This indicates that the early increase of viral oncogene expression is crucial for induction of papillomatosis.

Skin tumor formation in human papillomavirus 8 transgenic mice is associated with a deregulation of oncogenic miRNAs and their tumor suppressive targets.

BACKGROUND Dysregulation of microRNA (miRNA) expression is regularly found in various types of cancer and contributes to tumorigenic processes. However, little is known about miRNA expression in non-melanoma skin cancer in which a pathogenic role of beta human papillomaviruses (HPV) is discussed. A carcinogenic potential of beta HPV8 could be demonstrated in a transgenic mouse model, expressing all early genes of HPV8 (HPV8-CER). A single UVA/B-dose induced oncogene expression and led to papilloma growth within three weeks. OBJECTIVE Expression of miRNAs and their targets during HPV8-mediated tumor formation in mice. METHODS Skin of untreated or UV-irradiated wild-type and HPV8-CER mice was analyzed for miRNA expression and localization by qPCR and in situ hybridization. MiRNA target protein expression was analyzed by immunohistochemical staining. RESULTS Early steps in skin tumor formation in HPV8-CER mice were associated with an upregulation of the oncogenic miRNA-17-5p, -21 and -106a and a downregulation of the tumor-suppressive miRNA-155 and -206, which could be demonstrated by qPCR and in situ hybridization. The respective targets of miRNA-21 and -106a, the tumor suppressors PTEN, PDCD4 and Rb with their pivotal role in cell cycle regulation, apoptosis and proliferation were found to be downregulated. CONCLUSION This is the first report demonstrating that a cutaneous HPV type deregulates the expression of miRNAs. These deregulations are closely related to the UV-induced upregulation of HPV8 oncogene levels, which suggest a direct or indirect HPV8-specific effect on miRNA expression. These data presume that HPV8 interferes with the miRNA mediated gene regulation to induce tumorigenesis.

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

BackgroundThe 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 (β-PV) play an important role in accelerating the early stages of skin tumorigenesis.MethodsWe investigated the effects of UV induced DNA damage in murine models of β-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.ResultsSkin 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.ConclusionWe 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.

Human Papillomavirus Type 8 E6 Oncoprotein Inhibits Transcription of the PDZ Protein Syntenin-2

ABSTRACT 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.

Molecular Mechanisms of Human Papillomavirus Induced Skin Carcinogenesis

Infection of the cutaneous skin with human papillomaviruses (HPV) of genus betapapillomavirus (βHPV) is associated with the development of premalignant actinic keratoses and squamous cell carcinoma. Due to the higher viral loads of βHPVs in actinic keratoses than in cancerous lesions, it is currently discussed that these viruses play a carcinogenic role in cancer initiation. In vitro assays performed to characterize the cell transforming activities of high-risk HPV types of genus alphapapillomavirus have markedly contributed to the present knowledge on their oncogenic functions. However, these assays failed to detect oncogenic functions of βHPV early proteins. They were not suitable for investigations aiming to study the interactive role of βHPV positive epidermis with mesenchymal cells and the extracellular matrix. This review focuses on βHPV gene functions with special focus on oncogenic mechanisms that may be relevant for skin cancer development.

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

Infection with viruses of the genus Betapapillomavirus, β-human papillomaviruses (β-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 β-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 β-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 β-catenin was elevated, no signs of β-catenin activity within the canonical Wnt signalling pathway could be detected. The upregulation of β-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 β-catenin and ZO-1 are direct targets of E7 of the oncogenic β-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.

Lack of integrin β5 in Merkel cell carcinomas and derived cell lines is frequently associated with Merkel cell polyomavirus positivity.

To the editor: Merkel cell carcinoma (MCC) is a rare but aggressive neuroendocrine skin neoplasia. The merkel cell polyomavirus (MCPyV), a double-stranded DNA tumor virus was discovered in 2008. It is found in approximately 80% of human MCC and is suspected to play a role in MCC development [1]. Both, small tumor and the aminoterminal moiety of the large tumor antigen, were found to be expressed in MCPyV-positive MCC [2]. Merkel cells are usually scattered in the basal cell layer of the epidermis along the dermo-epidermal junction [3]. A number of MCC cell lines, with and without clonally integrated MCPyV, have been established from MCC tumours and represent valuable tools to study aspects of tumourigenesis in vitro. The MCPyV-negative cell lines UISO [4], MCC13 [5] and MCC26 [6] grow adhesively, whereas MaTi cells [7] grow in suspension. The MCPyV-positive cells MKL-1, MS-1, WaGa [2] and MKL-2 [6] all grow in suspension. Cells growing in suspension have not only lost their capability to bind to the extracellular matrix (ECM), but also proliferate slower than those growing adherently [2, 7]. Cells of all types interact with the ECM via integrins, heterodimeric transmembrane receptors composed by an α and β subunit. Integrins not only participate in cell-ECM adhesion, but may also function as receptors that transduce signals to the cell interior via the cytoskeleton and thus stimulate proliferation [8]. We therefore hypothesized that non-adherent MCC cell lines may show differences in integrin expression compared to adherent MCC cell lines, which are responsible for attachment and their growth behaviour. To prove this, we evaluated the expression of integrin subunits α2, α3, α5, α6, αv, β1, β3, β4, β5 and β6, known to be expressed in the human skin [8], on the adherent and non-adherent MCC cell lines. Samples were analyzed in duplicate together with a dilution series of a standard-plasmid containing the corresponding cDNA, which was used to generate a standard curve. To emphasize the relative differences between subunits, the mRNA expression levels were calculated relative to the absolute amounts of Hypoxanthin-Phosphoribosyl-Transferase-1 (HPRT1) transcripts used as internal control. The expression of integrin subunits was initially measured in the adherent MCPyV-negative line UISO and the MCPyV-positive line MS-1, growing in suspension. The qRT-PCR results revealed that both cell lines do not express integrin α2, α5, α6, β3, β4 and β6 (data not shown). The integrins αv and β1 are expressed in comparable amounts by both UISO and MS-1 cells whereas transcript levels of integrin α3 and β5 were high in UISO cells and barely detectable in MS-1 cells, with a 96- and 2180-fold difference respectively (Figure 1a). To confirm this observation at the protein level Western blots of cell extracts were evaluated. In line with qRT-PCR results, α3 and β5 proteins were highly expressed in UISO cells but undetectable in MS-1 cells (Figure 1b). Figure 1 (A) Integrin mRNA expression in UISO and MS-1 cells was quantified in qRT-PCR and normalized to the expression level of HPRT1. (B, C) Total cell extracts were prepared with RIPA buffer and analysed by Western blotting. Blots were probed with antibodies ... We next studied the expression of α3- and β5-integrin levels on additional MCPyV-positive and MCPyV-negative cell lines using whole cell lysates. As shown in Figure 1c, the expression level of β5-integrin was also high in the adherent MCPyV-negative cell lines MCC13 and MCC26 but low in MaTi cells, which grow in suspension. In the MCPyV-positive lines MKL-1, MKL-2 and WaGa, all growing in suspension, β5-integrin was undetectable. In all these cell lines, α3-integrin could not be detected. The expression of α3-integrin in UISO cells thus appears to be exceptional and lack of β5-integrin expression correlates with the MCC cell line growth in suspension. This does not exclude that other transmembrane molecules contribute to cell adhesion. This phenotype is also rather strongly correlated with MCPyV positivity (Table S1). We were next interested to study β5-integrin expression in MCPyV-positive and -negative MCC in vivo and therefore performed immunhistochemical staining on paraffin embedded sections. Nine MCC cases were obtained from the files of the Department of Dermatology and Venerology of the University of Cologne. MCPyV load quantification has been performed as previously described [9]. For virus-negative MCC, three showed strong (Fig. 2A) and two showed intermediate β5-integrin staining. For MCPyV-positive MCC, one tumor with a load of 2.6 viral DNA copies per cell had intermediate β5-integrin staining (Fig. 2B) while the remaining three tumors (one with a viral load of 2.8 MCPyV DNA copies per cell and two with 23 copies per cell) showed weak staining for β5-integrin (Fig. 2C). Thus, viral positivity inversely correlated with β5-integrin expression. Figure 2 Formalin fixed and paraffin-embedded MCC sections were stained for β5-integrin and counterstained with hematoxylin. MCC with high (A), medium (B) and weak (C) β5-integrin staining are shown. Magnification: 200x. This data provides some explanation, why MCC cell lines show distinct growth behaviour in culture related to MCPyV status. In vivo MCPyV-positive MCC are more likely to have low β5-integrin, which is characteristic for MCC cell lines growing in suspension. In adherent cells the presence of β5 and αv subunits can lead to formation of the vitronectin receptor αvβ5, which may be important for attachment and migration of Merkel cells. Changes in cell-matrix adhesion may affect metastasis formation. Whether, in addition to viral positivity a progressive loss of β5-integrin expression influences MCC prognosis is presently not clear. Additional studies are needed to clarify, if MCPyV directly or indirectly affects β5-integrin expression and thereby growth and attachment of Merkel cells.