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To evaluate the expression of lincRNA-p21, H19, EMX2OS, SNHG12 and MALAT1 in a mouse model of human papillomavirus 16 (HPV16)-induced carcinogenesis and cachexia.Chest skin, ear, tongue, penis and gastrocnemius muscle samples from wild-type mice (HPV-) and K14-HPV16 male mice (HPV+) were collected to evaluate the expression of the selected lncRNAs using real-time PCR (qPCR).In chest skin and ear, H19, SNHG12, EMX2OS and lincRNA-p21 were down-regulated in HPV+ versus HPV- mice. In tongue and penile tissues, there was only down-regulation of lincRNA-p21 in HPV+ mice. Additionally, in penile tissue, lincRNA-p21 expression decreased in HPV-induced lesions comparing with normal tissues. In gastrocnemius muscle, MALAT1 was up-regulated and lincRNA-p21 was down-regulated in HPV+ versus HPV-mice.H19, SNHG12, EMX2OS and lincRNA-p21 may be involved in HPV-induced carcinogenesis. In addition, MALAT1 and lincRNA-p21 may play a role in muscle wasting and contribute to cancer cachexia.

Simple Summary Colorectal cancer is an increasingly prevalent disease that accounts for substantial mortality and morbidity and is responsible for an impaired quality of life. This scenario highlights the urgent need to better understand the biological mechanisms underlying colorectal cancer onset, progression and spread to improve diagnosis and establish tailored therapeutic strategies. Therefore, understanding tumor microenvironment dynamics could be crucial, since it is where the tumorigenic process begins and evolves under the heavy influence of the complex crosstalk between all elements: the cellular component (cancer cells and the non-malignant stromal cells), the non-cellular component (extracellular matrix) and the interstitial fluids. Bioengineered models that can accurately mimic the tumor microenvironment are the golden key to comprehending disease biology. Therefore, the focus of this review addresses the advanced 3D-based models of the decellularized extracellular matrix as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro two-dimensional and in vivo models. Abstract More than a physical structure providing support to tissues, the extracellular matrix (ECM) is a complex and dynamic network of macromolecules that modulates the behavior of both cancer cells and associated stromal cells of the tumor microenvironment (TME). Over the last few years, several efforts have been made to develop new models that accurately mimic the interconnections within the TME and specifically the biomechanical and biomolecular complexity of the tumor ECM. Particularly in colorectal cancer, the ECM is highly remodeled and disorganized and constitutes a key component that affects cancer hallmarks, such as cell differentiation, proliferation, angiogenesis, invasion and metastasis. Therefore, several scaffolds produced from natural and/or synthetic polymers and ceramics have been used in 3D biomimetic strategies for colorectal cancer research. Nevertheless, decellularized ECM from colorectal tumors is a unique model that offers the maintenance of native ECM architecture and molecular composition. This review will focus on innovative and advanced 3D-based models of decellularized ECM as high-throughput strategies in colorectal cancer research that potentially fill some of the gaps between in vitro 2D and in vivo models. Our aim is to highlight the need for strategies that accurately mimic the TME for precision medicine and for studying the pathophysiology of the disease.

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.

Human papillomavirus (HPV) is the most common sexually transmitted infectious agent worldwide, being also responsible for 5% of all human cancers. The integration and hypermethylation mechanisms of the HPV viral genome promote the unbalanced expression of the E6, E7 and E5 oncoproteins, which are crucial factors for the carcinogenic cascade in HPV-induced cancers. This review highlights the action of E6, E7 and E5 over key regulatory targets, promoting all known hallmarks of cancer. Both well-characterized and novel targets of these HPV oncoproteins are described, detailing their mechanisms of action. Finally, this review approaches the possibility of targeting E6, E7 and E5 for therapeutic applications in the context of cancer.

Bracken (Pteridium spp.) is a common weed that is consumed as food especially in Asia, and is suspected of promoting carcinogenesis induced by papillomaviruses in the digestive and urinary systems. This is particularly worrying because the incidence of head-and-neck cancers associated with the human papillomavirus (HPV) is rapidly increasing, and HPV co-carcinogens urgently need to be identified. This study tested the hypothesis that two bracken compounds, ptaquiloside and rutin, are able to promote head-and-neck and bladder carcinogenesis in HPV16-transgenic mice. Expression of HPV16 E6 and E7 in oral and bladder tissues was confirmed using quantitative real-time PCR. Mice were exposed orally to ptaquiloside (0.5 mg per animal per week for 10 weeks from 20 weeks-old) or rutin (413 mg kg-1 day-1 for 24 weeks from 6 weeks-old), sacrificed at 30 weeks-old and studied histologically. HPV16 E6 and E7 expression was higher in oral mucosa compared with the bladder (p 0.001). Importantly, ptaquiloside, but not rutin, increased the incidence of oral squamous cell carcinomas (p = 1.2 × 10-8) in HPV16-transgenic mice. Also, cancers of unexposed transgenic mice were restricted to the tongue base, while ptaquiloside-exposed mice showed multifocal lesions throughout the oral cavity. Wild-type controls showed no oral lesions. No bladder lesions were observed in any treated or untreated group. These results indicate that ptaquiloside from bracken is able to promote oral carcinogenesis initiated by HPV16. Rutin did not show any carcinogenic effects in this model. The absence of bladder lesions may reflect an insufficient incubation period or factors related to the specific viral oncogenes present in this model.

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.

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.

The CRISPR-Cas9, a part of the defence mechanism from bacteria, has rapidly become the simplest, fastest and the most precise genome-editing tool available. The therapeutic applications of CRISPR are boundless: correction of mutations in several disorders, inactivation of oncogenes and viral oncoproteins, and activation of tumor suppressor genes. In this review, we expose recent advances concerning animal models of cancer that use CRISPR-Cas9, addressing also the current efforts to develop CRISPR-Cas9-based therapies, focusing on proof-of-concept studies. Finally, the review exposes some of the main challenges that this genome-editing tool faces. The key issue remains: does CRISPR-Cas9 have real potential for therapeutic application or will it just remain a wonderful research tool?

Tese de mestrado, Física (Física da Matéria Condensada e Nano-materiais) Universidade de Lisboa, Faculdade de Ciências, 2017 Submitted by Teresa Boa (tdboa@fc.ul.pt) on 2017-05-11T10:17:02Z No. of bitstreams: 1 ulfc121660_tm_Diogo_Pinto.pdf: 2242923 bytes, checksum: 29b7abaf9d4c09513d31699c88b73007 (MD5) Made available in DSpace on 2017-05-11T10:17:10Z (GMT). No. of bitstreams: 1 ulfc121660_tm_Diogo_Pinto.pdf: 2242923 bytes, checksum: 29b7abaf9d4c09513d31699c88b73007 (MD5) Previous issue date: 2017