HPV16 induces penile intraepithelial neoplasia and squamous cell carcinoma in transgenic mice: first mouse model for HPV-related penile cancer.

Penile cancer is an under-studied disease that occurs more commonly in developing countries and 30-50% of cases show high-risk human papillomavirus (HPV) infection. Therapeutic advances are slow, largely due to the absence of animal models for translational research. Here, we report the first mouse model for HPV-related penile cancer. Ten-week-old mice expressing all the HPV16 early genes under control of the cytokeratin 14 (Krt14) gene promoter and matched wild-type controls were exposed topically to dimethylbenz(a)anthracene (DMBA) or vehicle for 16 weeks. At 30 weeks of age, mice were sacrificed for histological analysis. Expression of Ki67, cytokeratin 14, and of the HPV16 oncogenes E6 and E7 was confirmed using immunohistochemistry and quantitative PCR, respectively. HPV16-transgenic mice developed intraepithelial lesions including condylomas and penile intraepithelial neoplasia (PeIN). Lesions expressed cytokeratin 14 and the HPV16 oncogenes E6 and E7 and showed deregulated cell proliferation, demonstrated by Ki67-positive supra-basal cells. HPV16-transgenic mice exposed to DMBA showed increased PeIN incidence and squamous cell carcinoma. Malignant lesions showed varied histological features closely resembling those of HPV-associated human penile cancers. Wild-type mice showed no malignant or pre-malignant lesions even when exposed to DMBA. These observations provide the first experimental evidence to support the etiological role of HPV16 in penile carcinogenesis. Importantly, this is the first mouse model to recapitulate key steps of HPV-related penile carcinogenesis and to reproduce morphological and molecular features of human penile cancer, providing a unique in vivo tool for studying its biology and advancing basic and translational research. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

HPV16 is sufficient to induce squamous cell carcinoma specifically in the tongue base in transgenic mice.

Head and neck squamous cell carcinomas (HNSCCs) associated with human papillomavirus (HPV) occur specifically in the tonsils and the tongue base, but the reasons for this specificity remain unknown. We studied the distribution of oral and pharyngeal lesions in HPV16-transgenic mice where the expression of all the HPV16 early genes is targeted to keratinising squamous epithelia by the cytokeratin 14 (Krt14) gene promoter. At 30 weeks of age, 100% of mice developed low- and high-grade intraepithelial dysplasia at multiple sites. Twenty per cent of animals developed invasive cancers that remarkably were restricted to the tongue base, in association with the circumvallate papilla. The lesions maintained expression of CK14 (KRT14) and the HPV16 E6 and E7 oncogenes, and displayed deregulated cell proliferation and up-regulation of p16INK4A . Malignant lesions were poorly differentiated and destroyed the tongue musculature. We hypothesised that the tongue base area might contain a transformation zone similar to those observed in the cervix and anus, explaining why HPV-positive cancers target that area specifically. Immunohistochemistry for two transformation zone markers, CK7 (KRT7) and p63 (TP63), revealed a squamocolumnar junction in the terminal duct of von Ebner's gland, composed of CK7+ luminal cells and p63+ basal cells. Dysplastic and invasive lesions retained diffuse p63 expression but only scattered positivity for CK7. Site-specific HPV-induced carcinogenesis in the tongue base may be explained by the presence of a transformation zone in the circumvallate papilla. This mouse model reproduces key morphological and molecular features of HPV-positive HNSCC, providing a unique in vivo tool for basic and translational research. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Dimethylaminoparthenolide reduces the incidence of dysplasia and ameliorates a wasting syndrome in HPV16-transgenic mice.

The nuclear factor kappa light chain enhancer of activated B cells (NF-κB) has been implicated in the progression of cancers induced by high-risk human papillomaviruses (HPV). In cancer patients, NF-κB is also thought to drive a chronic systemic inflammatory status, leading to cachexia. This study addressed the ability of dimethylaminoparthenolide (DMAPT), a water-soluble NF-κB inhibitor, to block the development of HPV-induced lesions and wasting syndrome in HPV16-transgenic mice. Mice received DMAPT orally (100 mg/kg/day), once a day, for 6 consecutive weeks. Body weight was monitored weekly along with food and water intake. After 6 weeks the animals were submitted to a grip strength test and sacrificed for specimen collection. Skin samples were analyzed histologically and for expression of NF-κB-regulated genes Bcl2 and Bcl2l1. Gastrocnemius muscles were weighted and analyzed for expression of NF-κB subunits p50, p52, p65, and Rel-B. DMAPT reduced the incidence of epidermal dysplasia (18.2% versus 33.3% in HPV16+/- untreated mice). This was associated with reduced expression of Bcl2 and Bcl2l1 (p = .0003 and p = .0014, respectively) and reduced neutrophilic infiltration (p = .0339). Treated mice also showed partially preserved bodyweight and strength, which were independent of the expression levels of NF-κB subunits in skeletal muscle.These results suggest that NF-κB inhibition may be a valid strategy against HPV-induced lesions in vivo and warrant further preclinical tests particularly in the set of combination therapies. In addition, the data may support the use of DMAPT to prevent wasting syndrome.

Curcumin and Rutin Down-regulate Cyclooxygenase-2 and Reduce Tumor-associated Inflammation in HPV16-Transgenic Mice.

AIM: Cyclo-oxygenase-2 (COX2) plays a prominent role in carcinogenesis. This study addresses the effects of two nutraceutical compounds on the expression of COX2 and tumor-associated inflammation in human papillomavirus type 16 (HPV16)-transgenic mice. MATERIALS AND METHODS: Six-week-old FVB/n mice were supplemented with rutin or curcumin for 24 weeks: HPV16-/- no treatment, n=12; HPV16+/- no treatment, n=13; HPV16+/- rutin, n=12; HPV16+/- curcumin, n=13. HPV16-induced skin lesions and their inflammatory infiltrates were studied histologically. COX2 expression was assessed immunohistochemically. RESULTS: Rutin reduced COX2 expression in the dermis (immunostaining score 7.83 versus 11.25 in untreated HPV16-transgenic mice) and epidermis (4.5 versus 10.0). Curcumin led to dermal and epidermal scores of 10.5 and 4.5. Both compounds reduced leukocytic infiltration, but neither prevented epidermal dysplasia. CONCLUSION: COX2 expression in HPV16-induced lesions may be modulated by nutraceuticals, reducing tumor-associated inflammation. However, this was not sufficient to block carcinogenesis, calling for additional studies focused on combination therapies.

Expression profile of microRNA-146a along HPV-induced multistep carcinogenesis: a study in HPV16 transgenic mice.

PURPOSE: Persistent human papillomavirus (HPV) infection is associated with the development of certain types of cancer and the dysregulation of microRNAs has been implicated in HPV-associated carcinogenesis. This is the case of microRNA-146a (miR-146a), which is thought to regulate tumor-associated inflammation. We sought to investigate the expression levels of miR-146a during HPV16-mediated carcinogenesis using skin samples from K14-HPV16 transgenic mice which develop the consecutive phases of the carcinogenesis process. METHODS: Female transgenic (HPV+/-) and wild-type (HPV-/-) mice were sacrificed at 24-26 weeks-old or 28-30 weeks-old. Chest and ear skin samples from HPV+/- and HPV-/- mice were histologically classified and used for microRNA extraction and quantification by qPCR. RESULTS: Chest skin samples from 24 to 26 weeks-old HPV+/- mice presented diffuse epidermal hyperplasia and only 22.5% showed multifocal dysplasia, while at 28-30 weeks-old all (100.0%) HPV+/- animals showed epidermal dysplasia. All HPV+/- ear skin samples showed carcinoma in situ (CIS). MiR-146a expression levels were higher in HPV+/- compared to HPV-/- mice (p = 0.006). There was also an increase in miR-146a expression in dysplastic skin lesions compared with hyperplasic lesions (p = 0.011). Samples showing CIS had a significant decrease in miR-146a expression when compared to samples showing epidermal hyperplasia (p = 0.018) and epidermal dysplasia (p = 0.009). CONCLUSIONS: These results suggest that HPV16 induces the overexpression of miR-146a in the initial stages of carcinogenesis (hyperplasia and dysplasia), whereas decreases its expression at later stages (CIS). Taken together, these data implicate and suggest different roles of miR-146a in HPV-mediated carcinogenesis.

Regulation of miRNA-146a and miRNA-150 Levels by Celecoxib in Premalignant Lesions of K14-HPV16 Mice.

BACKGROUND/AIM: Human papillomavirus type 16 (HPV16) induces various types of cancer in several locations. Microenvironmental microRNAs (miRNAs) such as miRNA-146a and miRNA-150 regulate cancer-associated inflammation and are involved in HPV-induced carcinogenesis. We studied the effects of celecoxib on the expression of these two miRNAs in HPV16-induced lesions. MATERIALS AND METHODS: Female transgenic (HPV16+/-) and wild-type (HPV16-/-) mice were administered 75 mg/kg/day celecoxib orally (treatment groups) or placebo (control groups) for four weeks. Skin samples were classified histologically, or used for miRNA analysis by quantitative real-time PCR. RESULTS: HPV16+/- mice showed higher miRNA-146a and miRNA-150 expression levels compared to wild-type animals. Celecoxib further increased miRNA-150 (p<0.05) and miRNA-146a levels in treated animals. Celecoxib-treated HPV16+/- animals also showed reduced incidence of epidermal dysplasia and reduced inflammation, compared to untreated mice. CONCLUSION: In this model, celecoxib may be able to regulate tumour-associated inflammation, through mechanisms involving the regulation of miRNA-146a and miRNA-150.

Dysregulated expression of microRNA-150 in human papillomavirus-induced lesions of K14-HPV16 transgenic mice.

AIMS: High-risk human papillomavirus (HPV) infection is one of the major causes of infection-related cancers worldwide. MicroRNAs (miRNAs) are a family of non-coding RNAs (ncRNAs), whose dysregulated levels may cause an aberrant expression of genes involved in oncogenic pathways and consequently lead to cancer development. This is the case of the miRNA-150 (miR-150), whose expression in HPV-induced lesions remains unclear and the present work aims to clarify it. We employed K14-HPV16 mice, which express the early genes of HPV16 in basal keratinocytes, leading to the development of hyperplastic and dysplastic skin lesions and squamous cell carcinomas, and are a representative model of HPV-induced cancers. MAIN METHODS: In order to evaluate the expression of miR-150 in HPV-induced lesions, we performed qPCR in wild-type mice (HPV-/-) and in skin lesions of K14-HPV16 transgenic mice (HPV+/-). Matched skin samples were analyzed histologically. KEY FINDINGS: 24-26weeks-old HPV+/- mice showed diffuse epidermal hyperplasia and focal dysplasia in a hyperplastic background (31.8% incidence), but 28-30weeks-old HPV+/- mice presented higher incidence of dysplasia (100.0%). MiR-150 was upregulated in HPV+/- mice when compared with HPV-/- mice (p<0.001). MiR-150 was also overexpressed in diffuse dysplastic lesions when compared with hyperplastic lesions (p=0.005). SIGNIFICANCE: The present results suggest that miR-150 is overexpressed in HPV-induced lesions in this model and its expression seems to increase with lesion progression, along the process of multi-step carcinogenesis.

HPV16 induces a wasting syndrome in transgenic mice: Amelioration by dietary polyphenols via NF-κB inhibition.

Cancer patients often show a wasting syndrome for which there are little therapeutic options. Dietary polyphenols have been proposed for treating this syndrome, but their usefulness in cases associated with human papillomavirus (HPV)-induced cancers is unknown. We characterized HPV16-transgenic mice as a model of cancer cachexia and tested the efficacy of long-term oral supplementation with polyphenols curcumin and rutin. Both compounds were orally administered to six weeks-old HPV16-transgenic mice showing characteristic multi-step skin carcinogenesis, for 24weeks. Skin lesions and blood, liver and spleen inflammatory changes were characterized histologically and hematologically. Hepatic oxidative stress, skeletal muscle mass and the levels of muscle pro-inflammatory transcription factor NF-κB were also assessed. Skin carcinogenesis was associated with progressive, severe, systemic inflammation (leukocytosis, hepatitis, splenitis), significant mortality and cachexia. Curcumin and rutin totally suppressed mortality while reducing white blood cells and the incidence of splenitis and hepatitis. Rutin prevented muscle wasting more effectively than curcumin. Preservation of muscle mass and reduced hepatic inflammation were associated with down-regulation of the NF-κB canonical pathway and with reduced oxidative stress, respectively. These results point out HPV16-transgenic mice as a useful model for studying the wasting syndrome associated with HPV-induced cancers. Dietary NF-κB inhibitors may be useful resources for treating this syndrome.