Analyse des onkogenen Potentials der frühen Gene E6 und E7 des humanen Papillomvirus Typ 8 durch die Etablierung transgener Mausmodelle.

Das kutane humane Papillomvirus 8 (HPV8) gehört zu den Hoch-Risiko Typen bei Epidermodysplasia verruciformis (EV)-Patienten. Mit Hilfe sensitiver Detektionstechniken konnte dieser EV-HPV auch in gesunder Haut und in nicht-melanozytärem Hautkrebs der Normalbevölkerung gefunden werden, vor allem bei immunsupprimierten Organ Transplantatempfängern. Durch die Etablierung transgener Mäuse mit der gesamten frühen Region (GFR) von HPV8 unter der Kontrolle des humanen Keratin-14 Promotors, welcher die Expression dieser Gene in basalen Hautkeratinozyten aktiviert, konnten wir das onkogene Potential von HPV8 in vivo demonstrieren. Da im HPV8-GFR Mausmodell alle frühen Gene von HPV8 gleichzeitig exprimiert werden, sollten im Rahmen dieser Dissertation die Gene E6 und E7 einzeln im transgenen Mausmodell untersucht werden, um deren Rolle in der Karzinogenese zu charakterisieren. Dabei stellte sich heraus, dass das E6 Protein alleine in der Lage ist singuläre oder multifokale gutartige Tumoren spontan zu induzieren. Diese Tumoren sind durch Papillomatose, Akanthose, Hyperkeratose und unterschiedliche Grade epidermaler Dysplasie gekennzeichnet und entarten in 6% der Fälle zu Plattenepithelkarzinomen, vergleichbar mit den Ergebnissen der HPV8-GFR Mäuse. Im Gegensatz dazu zeigten die HPV8-E7 transgenen Mäuse keinen offensichtlichen Phänotyp. Um synergistische Effekte des UV-Lichtes und der Wundheilung in diesen transgenen Tieren zu evaluieren, wurde die Haut der HPV8 Mäuse mit UVA und UVB bestrahlt oder mit Biopsiestanzen verwundet. Die UVA/B Bestrahlung und die Verwundung induzierten in den HPV8-GFR und -E6 transgenen Mäusen zwei bis drei Wochen nach der Behandlung Papillomatose. Die Bestrahlung mit UVA hatte keine induzierende Wirkung und die Bestrahlung mit UVB alleine erwies sich als schwacher Induktor der Papillomatose. In 40% der untersuchten PEK konnten Mutationen im Codon 61 des H-ras Gens identifiziert werden. Mutationen im p53 Gen wurden weder in benignen noch in malignen Tumoren von HPV8 positiven Mäusen gefunden. Immunhistochemische Färbungen von p53 zeigten gleiche Spiegel von p53 in der Epidermis HPV8 positiver und negativer Mäuse nach UV Bestrahlung. In der murinen Epidermis ist das E6 Protein von HPV8 das Hauptonkogen, notwendig und ausreichend, um eine spontane Tumorentwicklung bis hin zum Karzinom zu induzieren. Eine HPV8 Infektion, die über Dekaden, obgleich auf einem niedrigen Niveau, fortbesteht, könnte in Interaktion mit Prozessen der Wundheilung, eine relevante Ursache von Hautkarzinogenese beim Menschen sein

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

Simple Summary Human papillomavirus (HPV) of genus beta (betaHPV) infects cutaneous epithelia and contributes to skin carcinogenesis, particularly in immunosuppressed patients. The HPV8 transgenic mouse model serves as a model for betaHPV-induced skin tumorigenesis. These animals express the complete early genome region of HPV8 under the control of the keratin (K)-14 promoter (K14-HPV8-CER). Skin tumorigenesis in these mice is driven by Lrig1-positive stem cells. To understand the role of HPV8 gene expression in Lrig1+ keratinocytes, we determined the transcriptional network in K14-HPV8-CER skin tumours and compared it to the already known pattern in Lrig1 stem cells. We showed that HPV8 differentially regulates 397 cellular genes in skin tumours and subverts the expression pattern of 23 genes in Lrig1+ cells. This study identified gene targets of HPV8 and its upstream regulators, which may play an important role in HPV8 mediated 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

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

Activation-induced cytidine deaminase is dispensable for virus-mediated liver and skin tumor development in mouse models

Activation-induced cytidine deaminase (AID) not only promotes immune diversity by initiating somatic hypermutation and class switch recombination in immunoglobulin genes but also provokes genomic instability by introducing translocations and mutations into non-immunoglobulin genes. To test whether AID is essential for virus-induced tumor development, we used two transgenic tumor models: mice expressing hepatitis C virus (HCV) core proteins (HCV-Tg), driven by the hepatitis B virus promoter, and mice expressing human papillomavirus type 8 proteins (HPV8-Tg), driven by the Keratin 14 promoter. Both strains were analyzed in the absence and presence of AID by crossing each with AID(-/-) mice. There was no difference in the liver tumor frequency between the HCV-Tg/AID(+/+) and HCV-Tg/AID(-/-) mice at 20 months of age although the AID(+/+) mice showed more severe histological findings and increased cytokine expression. Furthermore, a low level of AID transcript was detected in the HCV-Tg/AID(+/+) liver tissue that was not derived from hepatocytes themselves but from intra-hepatic immune cells. Although AID may not be the direct cause of HCV-induced oncogenesis, AID expressed in B cells, not in hepatocytes, may prolong steatosis and cause increased lymphocyte infiltration into HCV core protein-induced liver lesions. Similarly, there was no difference in the time course of skin tumor development between the HPV8-Tg/AID(-/-) and HPV8-Tg/AID(+/+) groups. In conclusion, AID does not appear to be required for tumor development in the two virus-induced tumor mouse models tested although AID expressed in infiltrating B cells may promote inflammatory reactions in HCV core protein-induced liver pathogenesis

ATP synthase modulation leads to an increase of spare respiratory capacity in HPV associated cancers

Mucosal and skin cancers are associated with infections by human papillomaviruses (HPV). The manner how viral oncoproteins hijack the host cell metabolism to meet their own energy demands and how this may contribute to tumorigenesis is poorly understood. We now show that the HPV oncoprotein E7 of HPV8, HPV11 and HPV16 directly interact with the beta subunit of the mitochondrial ATP-synthase (ATP5B), which may therefore represent a conserved feature across different HPV genera. By measuring both glycolytic and mitochondrial activity we observed that the association of E7 with ATP5B was accompanied by reduction of glycolytic activity. Interestingly, there was a drastic increase in spare mitochondrial respiratory capacity in HPV8-E7 and an even more profound increase in HPV16-E7 expressing cells. In addition, we could show that ATP5B levels were unchanged in betaHPV positive skin cancers. However, comparing HPV-positive and HPV-negative oropharyngeal squamous cell carcinomas (OPSCC) we noticed that, while ATP5B expression levels did not correlate with patient overall survival in HPV-negative OPSCC, there was a strong correlation within the HPV16-positive OPSCC patient group. These novel findings provide evidence that HPV targets the host cell energy metabolism important for viral life cycle and HPV-mediated tumorigenesis