The interplay of UV and cutaneous papillomavirus infection in skin cancer development.

Cutaneous human papillomaviruses (HPVs) are considered as cofactors for non-melanoma skin cancer (NMSC) development, especially in association with UVB. Extensively studied transgenic mouse models failed to mimic all aspects of virus-host interactions starting from primary infection to the appearance of a tumor. Using the natural model Mastomys coucha, which reflects the human situation in many aspects, we provide the first evidence that only UVB and Mastomys natalensis papillomavirus (MnPV) infection strongly promote NMSC formation. Using UVB exposures that correspond to UV indices of different geographical regions, irradiated animals developed either well-differentiated keratinizing squamous cell carcinomas (SCCs), still supporting productive infections with high viral loads and transcriptional activity, or poorly differentiated non-keratinizing SCCs almost lacking MnPV DNA and in turn, early and late viral transcription. Intriguingly, animals with the latter phenotype, however, still showed strong seropositivity, clearly verifying a preceding MnPV infection. Of note, the mere presence of MnPV could induce γH2AX foci, indicating that viral infection without prior UVB exposure can already perturb genome stability of the host cell. Moreover, as shown both under in vitro and in vivo conditions, MnPV E6/E7 expression also attenuates the excision repair of cyclobutane pyrimidine dimers upon UVB irradiation, suggesting a viral impact on the DNA damage response. While mutations of Ras family members (e.g. Hras, Kras, and Nras) were absent, the majority of SCCs harbored-like in humans-Trp53 mutations especially at two hot-spots in the DNA-binding domain, resulting in a loss of function that favored tumor dedifferentiation, counter-selective for viral maintenance. Such a constellation provides a reasonable explanation for making continuous viral presence dispensable during skin carcinogenesis as observed in patients with NMSC

Summary of the absolute numbers of tumors in the different groups, percentage of tumor-bearing animals and median time of tumor development.

<p>Summary of the absolute numbers of tumors in the different groups, percentage of tumor-bearing animals and median time of tumor development.</p

Molecular analyses of tumor-bearing animals.

<p><b>A)</b> Viral load in tissue samples from UV-irradiated and control animals from the MnPV-infected colony analyzed by qPCR and normalized to a plasmid standard. Samples were grouped according to their origin as indicated (ctrl skin: skin from unirradiated animals; ui skin/UV skin: unirradiated or UV-irradiated skin from irradiated animals; KSCC/nKSCC: UV-induced SCCs; non-UV tumor: tumors from non-UV sites of irradiated animals and spontaneous tumors from unirradiated animals). UV^+/- indicates whether the animal was UV-exposed or not (Kruskal-Wallis test, *p<0.05, ***p<0.001, ^nsp>0.05). <b>B)</b> Southern blot analysis of unirradiated and UV-irradiated skins, a KSCC and a non-UV tumor. DNA was digested with ApaI (no cleavage site in MnPV), XbaI (one site) or XhoI (two sites) as indicated (Form I: supercoiled; Form II: relaxed circular; Form III: linear form of MnPV). <b>C)</b> Semi-quantitative RT-PCR for the most abundant MnPV <i>E1^E4</i> transcript in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>. <b>D)</b> Semi-quantitative RT-PCR for MnPV <i>E6</i>, <i>E7</i> and <i>L1</i> transcripts in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>.</p

MnPV interferes with DNA damage repair.

<p><b>A)</b> Repair kinetics of CPDs in MnPV E6/E7-positive and -negative <i>Mastomys</i> keratinocytes (Mean ± SD; n = 2, measurements were performed in quadruplicates). <b>B)</b> Immunofluorescence staining of γH2AX foci in keratinocytes stably expressing MnPV E6/E7. Cells were irradiated with UVB and further incubated prior to detection and quantification of γH2AX foci (Ctrl: unirradiated, UV: irradiated; Red: γH2AX, blue: nuclei; scale bars: 50 μm). <b>C)</b> Quantification of γH2AX foci (Mean ± SEM; n≥242; 1way-ANOVA, *p<0.05, **p<0.01, ***p<0.001). <b>D)</b> Co-detection of CPDs and γH2AX in MnPV^+/- skin harvested 24h after UV irradiation. Arrows point towards positive cells (Viral loads: animal 3: 13.68 ± 1.66 copies/cell, animal 4: 147.42 ± 14.62 copies/cell; Scale bars: 100 μm). <b>E)</b> Co-detection of CPDs and γH2AX in a KSCC harvested 24h after UV irradiation (Viral load: 611.88 ± 18.75 copies/cell; scale bars: 100 μm).</p

Collagen IV staining on tissue sections reveals invasion of keratinocytes through the basal membrane (BM).

<p>The BM was stained against collagen IV (green). Nuclei were counterstained with DAPI (blue). Consecutive sections stained for pan-cytokeratin are shown in comparison. <b>A)</b> In normal skin, the BM (white arrows) marks the barrier between epidermis and dermis. <b>B)</b> Early stage carcinoma formation in UV-irradiated skin. A lack of collagen IV expression indicates the disruption of the BM (orange arrows) accompanied by downward migrating cells (black arrow). <b>C)</b> In the edge region of a UV-induced KSCC, the BM is lost and invading altered keratinocytes are detectable. <b>D)</b> In nKSCC, invasion of neoplastic cells is advanced as indicated by pan-cytokeratin staining. The discontinuous staining of the BM marks transition zones where invading neoplastic squamous cells acquire a spindle cell phenotype (Scale bars: 100 μm).</p

Spatial analysis of viral load in UV-induced SCCs.

<p>Quantification and distribution of MnPV DNA in different microdissected areas (specified in the HE staining) of <b>A)</b> a KSCC and <b>B)</b> a UV-induced nKSCC. Asterisks indicate the position of the respective MnPV-specific <i>in situ</i> hybridization (ISH) (Scale bars: HE: 1 mm, ISH: 100 μm).</p

Study design and tumor development.

<p><b>A)</b><i>Mastomys coucha</i> as a model for cutaneous papillomavirus infection. In the study, naturally MnPV-infected animals (MnPV⁺) as well as virus-free control animals (MnPV^-) were irradiated three times per week with UVB. The starting dose of 150 mJ/cm² was increased weekly by 50 mJ/cm² until the desired final dose was reached (450, 600 or 800 mJ/cm², respectively). Black arrows indicate an increase of the dose, gray arrows the subsequent application of this dose. The irradiation was continued until the animals were sacrificed or died. <b>B)</b> Kaplan-Meier curves demonstrating the percentage of irradiated virus-infected (MnPV⁺, UV⁺), virus-free (MnPV^-, UV⁺) and unirradiated virus-infected (MnPV⁺, UV^-) tumor-bearing animals. <b>C)</b> Two examples of spontaneous skin lesions arising in naturally infected animals. <b>D)</b> Examples of UV-induced keratinizing SCCs (KSCC) with similarities to human keratoacanthomas. <b>E)</b> Examples of UV-induced non-keratinizing SCCs (nKSCC) (C, D and E: scale bars: 10 mm). <b>F)</b> Number of KSCCs and nKSCCs in correlation with the final UV doses. Note that KSCCs preferentially appeared at the lowest dose, nKSCCs preferentially at higher doses (Mean ± SEM; animal numbers: see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006723#ppat.1006723.t001" target="_blank">Table 1</a>; av: average number of tumors).</p

Schematic overview of the mechanism suggested for UV-induced NMSC development in <i>Mastomys coucha</i>.

<p><b>A)</b> MnPV infects basal epithelial cells of the skin of young animals via small injuries. <b>B)</b> MnPV genome is amplified in stratified skin layers (pink and red nuclei) and new virions are released. <b>C)</b> UVB irradiation of the skin. <b>D)</b> UVB-irradiated skin is hyperproliferative, favoring viral replication and virion formation. UVB-induced photoproducts, e.g. in <i>Trp53</i>, occur in keratinocytes (altered nuclei). In uninfected cells, damages are repaired. In infected cells, MnPV-E6/E7 reduce chromosomal stability and inhibit DNA repair. Mutations can accumulate and altered cells become neoplastic. <b>E)</b> Neoplastic squamous cells (light blue) start forming a well-differentiated keratinizing SCC, still representing a permissive system that allows viral replication and formation of virions. <b>F)</b> When neoplastic squamous cells accumulate further mutations (dark blue), a spindle cell phenotype is acquired, forming a poorly differentiated SCC that may become ulcerated. MnPV cannot replicate in dedifferentiated cells and the viral DNA is subsequently lost.</p

Dedifferentiation correlates with positive p53 staining.

<p>Consecutive sections of a poorly differentiated nKSCC were stained with antibodies against E-cadherin, vimentin, pan-Cytokeratin and p53. DAPI was used as nuclear counter stain. Note that in this tumor, only mutation R266C could be detected (Scale bars: 100 μm).</p