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.

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.

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.

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.

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.

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.

Enhanced StefinA and Sprr2 expression during papilloma formation in HPV8 transgenic mice.

BACKGROUND: The human papillomavirus type 8 (HPV8) is associated with the development of non-melanoma skin cancer. Transgenic mice expressing the complete early gene region of HPV8 (E6/E7/E1/E2/E4=CER) or E6 separately under the control of the keratin14 promoter spontaneously developed papillomas characterized by varying degrees of epidermal dysplasia. Papilloma growth could be synchronized by a single UVA/B irradiation of the skin, which led to the development of papillomas within three weeks. OBJECTIVE: The objective of this study was to identify alterations in cellular gene expression correlated with HPV8 oncogene expression in transgenic mice. METHODS: We applied global gene expression profiling by microarray analysis and confirmed deregulation of cellular genes by qRT-PCR and immunohistochemical analysis. RESULTS: By comparison of non-lesional HPV8-CER skin with skin of the parental mouse strain FVB/n, two cellular genes, namely StefinA and Sprr2, coding for precursor proteins of the cornified envelope, were predicted to be strongly upregulated in transgenic skin, which could be confirmed in subsequent qRT-PCR experiments. StefinA and Sprr2 mRNA expression was enhanced until day 7 after UV treatment with higher levels in HPV8 positive skin. While the expression of both genes returned to a normal level in the course of epidermis regeneration in wt mice, the expression persisted elevated in hyperplastic transgenic skin. Staining of an UV induced papilloma of FVB/n wt mouse revealed also strong expression of StefinA and Sprr2 indicating that upregulation in later stages of papilloma formation is independent of HPV8. CONCLUSION: In non-lesional HPV8-CER transgenic skin StefinA and Sprr2 were found to be indirect/direct transcriptional targets of HPV8.

Keratinocyte-specific stat3 heterozygosity impairs development of skin tumors in human papillomavirus 8 transgenic mice.

Human papillomaviruses (HPV) of the genus ß are thought to play a role in human skin cancers, but this has been difficult to establish using epidemiologic approaches. To gain insight into the transforming activities of ß-HPV, transgenic mouse models have been generated that develop skin tumors. Recent evidence suggests a central role of signal transducer and activator of transcription 3 (Stat3) as a transcriptional node for cancer cell-autonomous initiation of a tumor-promoting gene signature associated with cell proliferation, cell survival, and angiogenesis. Moreover, high levels of phospho-Stat3 have been detected in tumors arising in HPV8-CER transgenic mice. In this study, we investigate the in vivo role of Stat3 in HPV8-induced skin carcinogenesis by combining our established experimental model of HPV8-induced skin cancer with epidermis-restricted Stat3 ablation. Stat3 heterozygous epidermis was less prone to tumorigenesis than wild-type epidermis. Three of the 23 (13%) Stat3(+/-):HPV8 animals developed tumors within 12 weeks of life, whereas 54.3% of Stat3(+/+):HPV8 mice already exhibited tumors in the same observation period (median age for tumor appearance, 10 weeks). The few tumors that arose in the Stat3(+/-):HPV8 mice were benign and never progressed to a more malignant phenotype. Collectively, these results offer direct evidence of a critical role for Stat3 in HPV8-driven epithelial carcinogenesis. Our findings imply that targeting Stat3 activity in keratinocytes may be a viable strategy to prevent and treat HPV-induced skin cancer.

The human papillomavirus type 8 E2 protein induces skin tumors in transgenic mice.

Transgenic mice expressing early genes of the cutaneous human papillomavirus 8 (HPV8) spontaneously develop skin papillomas, epidermal dysplasia, and squamous cell carcinoma (6%). As the HPV8 protein E2 revealed transforming capacity in vitro, we generated three epidermal specific HPV8-E2-transgenic FVB/N mouse lines to dissect its role in tumor development. The rate of tumor formation in the three lines correlated with the different E2-mRNA levels. More than 60% of heterozygous line 2 mice, but none of the HPV8-negative littermates, spontaneously developed ulcerous lesions of the skin over an observation period of up to 144 weeks, beginning on average 74+/-22 weeks after birth. Most lesions presented infundibular hyperplasia and acanthosis combined with low-grade dysplasia. Severe dysplasia of the epidermis occurred in 6%. Two carcinomas revealed a sharply demarcated spindle-cell component. Only 3 weeks after a single UV irradiation, 87% of heterozygous line 2 and 36% of line 35 mice developed skin tumors. A rapidly growing invasive tumor composed of spindle cells arose 10 weeks after irradiation of a line-35 animal. The histology of skin cancers in HPV8-E2 mice is reminiscent of a subset of highly aggressive squamous cell carcinoma in immunosuppressed transplant recipients with a massive spindle-cell component.

Expression of matrix metalloproteinase (MMP)-2, MMP-9, MMP-13, and MT1-MMP in skin tumors of human papillomavirus type 8 transgenic mice.

Human papillomaviruses (HPV) are small DNA viruses that induce a wide variety of hyperproliferative lesions in cutaneous and mucosal epithelia. It is proposed that HPV is involved in non-melanoma skin cancer development. We have previously shown that HPV8 transgenic mice spontaneously develop papillomatous skin tumors. Histology revealed epidermal hyperplasia, acanthosis and hypergranulosis and in some cases squamous cell carcinomas (SCC). Zymographic and immunoblot analysis of normal skin extracts identified increased amounts of matrix metalloproteinase (MMP)-9, MMP-13 and MT1-MMP in HPV8-positive mice compared with HPV8-negative animals. In situ gelatin zymography of tumor specimens displayed a strong proteolytic activity in papillomas, and SCC putatively attributed to the increased amounts of activated MMP-9 found in tissue extracts. In addition, immunoblot analysis revealed increased amounts of active MMP-13 and MT1-MMP in tumor extracts as compared with control extracts. Immunohistochemical stainings of SCC specimens depicted MMP-13 to be specifically expressed in stromal fibroblasts neighboring the tumor islands, whereas MT1-MMP was detected both in tumor cells and in stromal cells. Taken together, these results implicate a role for MMPs in the development of HPV8-induced cutaneous tumors.