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 p16 INK4A . 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.No conflicts of interest were declared. or HPV18, are associated with HNSCC cases occurring at specific oropharyngeal sitesthe tonsils and the tongue base. This site specificity of HPV-positive lesions suggests the presence of HPV-sensitive transformation zones, such as those described in the uterine cervix and the anus [2,3]. However, those areas, if they indeed exist, Journal of Pathology

Cancers induced by human papillomavirus (HPV) infection remain a significant public health threat, fueling the study of new therapies. Laurel (Laurus nobilis) compounds and extracts recently showed in vitro activity against HPV-transformed cell lines. This work aims to evaluate the in vivo efficacy and hepatic toxicity of a laurel extract in a transgenic mouse model of HPV16-induced cancer. The extract was administered in drinking water (20 mg per animal per day) for three consecutive weeks, using four experimental groups (n = 10) (group I: HPV16-/- without treatment, group II: treated HPV16-/-, group III: HPV16+/- without treatment and group IV: treated HPV16+/-). Following the treatment period, animals were sacrificed and skin samples were used to classify skin lesions histologically. Toxicological parameters included hematological and biochemical blood markers, splenic and hepatic histology and hepatic oxidative stress. The extract did not prevent the progression of HPV16-induced cutaneous lesions in this model. The treated wild-type animals showed mild hepatitis, while transgenic animals suffered weight loss. However, there were no changes concerning hematological, biochemical and hepatic oxidative stress markers.

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. Tenweek-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 HPVrelated 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.

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Cancer cachexia is a multifactorial syndrome characterized by general inflammation, weight loss and muscle wasting, partly mediated by ubiquitin ligases such as atrogin-1, encoded by Fbxo32. Cancers induced by high-risk human papillomavirus (HPV) include anogenital cancers and some head-and-neck cancers and are often associated with cachexia. The aim of this study was to assess the presence of cancer cachexia in HPV16-transgenic mice with or without exposure to the chemical carcinogen 7,12-dimethylbenz(a)anthracene (DMBA). Male mice expressing the HPV16 early region under the control of the cytokeratin 14 gene promoter (K14-HPV16; HPV+) and matched wild-type mice (HPV−) received DMBA (or vehicle) topically over 17 weeks of the experiment. Food intake and body weight were assessed weekly. The gastrocnemius weights and Fbxo32 expression levels were quantified at sacrifice time. HPV-16-associated lesions in different anatomic regions were classified histologically. Although unexposed HPV+ mice showed higher food intake than wild-type matched group (p < 0.01), they presented lower body weights (p < 0.05). This body weight trend was more pronounced when comparing DMBA-exposed groups (p < 0.01). The same pattern was observed in the gastrocnemius weights (between the unexposed groups: p < 0.05; between the exposed groups: p < 0.001). Importantly, DMBA reduced body and gastrocnemius weights (p < 0.01) when comparing the HPV+ groups. Moreover, the Fbxo32 gene was overexpressed in DMBA-exposed HPV+ compared to control mice (p < 0.05). These results show that K14-HPV16 mice closely reproduce the anatomic and molecular changes associated with cancer cachexia and may be a good model for preclinical studies concerning the pathogenesis of this syndrome.

A growing proportion of oropharyngeal squamous cell carcinomas (OPSCC) are associated with infection by high-risk human papillomavirus (HPV). For reasons that remain largely unknown, HPV+OPSCC is significantly more common in men than in women. This study aims to determine the incidence of OPSCC in male and female HPV16-transgenic mice and to explore the role of female sex hormone receptors in the sexual predisposition for HPV+ OPSCC. The tongues of 30-weeks-old HPV16-transgenic male (n = 80) and female (n = 90) and matched wild-type male (n = 10) and female (n = 10) FVB/n mice were screened histologically for intraepithelial and invasive lesions in 2017 at the Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Portugal. Expression of estrogen receptors alpha (ERα) and beta (ERβ), progesterone receptors (PR) and matrix metalloproteinase 2 (MMP2) was studied immunohistochemically. Collagen remodeling was studied using picrosirius red. Female mice showed robust ERα and ERβ expression in intraepithelial and invasive lesions, which was accompanied by strong MMP2 expression and marked collagen remodeling. Male mice showed minimal ERα, ERβ and MMP2 expression and unaltered collagen patterns. These results confirm the association of HPV16 with tongue base cancer in both sexes. The higher cancer incidence in female versus male mice contrasts with data from OPSCC patients and is associated with enhanced ER expression via MMP2 upregulation.