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.

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.

Prevalence and associated factors of betapapillomavirus infections in individuals without cutaneous squamous cell carcinoma.

Betapapillomavirus (betaPV) infections are often associated with squamous-cell carcinoma (SCC) and the prevalence of betaPV infections in (immunosuppressed) SCC patients is known to be high. The distribution and possible associated factors of betaPV infections in the general population, however, are largely unknown. To address this issue, betaPV infection was studied in 1405 SCC-free immunocompetent (n=845) and immunosuppressed (n=560) individuals from six countries of different latitudes. A standard study protocol was used to obtain information about age, sex, UV-irradiation and skin type, and from all participants eyebrow hairs were collected for detection and genotyping of 25 established betaPV types using the PM-PCR reverse hybridization assay (RHA) method. The frequency of betaPV-positive participants ranged from 84 to 91% in the immunocompetent population with HPV23 as the most prevalent type, and from 81 to 98% in the immunosuppressed population with HPV23 as the most or the second most prevalent type. The median number of infecting betaPV types ranged from four to six in the immunocompetent and from three to six in the immunosuppressed population. Increasing age in the immunocompetent participants and (duration of) immunosuppression in the immunosuppressed patients were associated with betaPV infection. In both groups, sex, skin phototype, sunburns and sun-exposure were not consistently associated with betaPV infection. This study demonstrates that betaPV infections are also highly prevalent in SCC-free individuals, with similar HPV types prevailing in both immunocompetent and immunosuppressed persons. Age and (duration of) immunosuppression were identified as betaPV infection-associated factors, whereas characteristics related to sun exposure and skin type were not.

Evaluation of a novel highly sensitive, broad-spectrum PCR-reverse hybridization assay for detection and identification of beta-papillomavirus DNA.

Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.

Development of skin tumors in mice transgenic for early genes of human papillomavirus type 8.

The cutaneous human papillomavirus (HPV) 8 is clearly involved in skin cancer development in epidermodysplasia verruciformis patients and its early genes E2, E6, and E7 have been implicated in cell transformation in vitro. To examine the functions of these genes in vivo we integrated the complete early region of HPV8 into the genome of DBA/Bl6 mice. To target their expression to the basal layer of the squamous epithelia the transgenes were put under the control of the keratin-14 promoter. Transgenic mice were back-crossed for up to six generations into both FVB/N and Bl6 mouse strains. Whereas none of the HPV8 transgene-negative littermates developed lesions in the skin or any other organ, 91% of HPV8-transgenic mice developed single or multifocal benign tumors, characterized by papillomatosis, acanthosis, hyperkeratosis, and varying degrees of epidermal dysplasia. Squamous cell carcinomas developed in 6% of the transgenic FVB/N mice. Real-time reverse transcription-PCR showed highest expression levels for HPV8-E2, followed by E7 and E6. There was no consistent difference in relative viral RNA levels between healthy or dysplastic skin and malignant skin tumors. Whereas UV-induced mutations in the tumor suppressor gene p53 are frequently detected in human skin carcinomas, mutations in p53 were not observed either in the benign or malignant mouse tumors. Nonmelanoma skin cancer developed in HPV8-transgenic mice without any treatment with physical or chemical carcinogens. This is the first experimental proof of the carcinogenic potential of an epidermodysplasia verruciformis-associated HPV-type in vivo.