Modelo de análisis in vivo de patologías asociadas a virus del papiloma humano oncogénicos

Abstract

Human papillomaviruses (HPVs) are DNA viruses of the Papillomaviridae family that infect stratified epithelia, and are responsible of benign and cancerous lesions in different anatomical locations. A few HPV types, considered as ¿high-risk¿ types (such as HPV 16 and 18), are the causative agents of virtually all cervical cancers, and some oropharyngeal carcinomas. Importantly, cervical cancer (CC) is the 3er most frequent cancer in women worldwide. Moreover, cutaneous HPV types belonging to the beta genus (such as HPV 5 and 8), are associated to squamous cell carcinoma (SCC) that frequently appeared in the skin of patients of the inherited skin disease Epidermodysplasia verruciformis, an also in organ transplant recipients. Analysis models for HPV-infection and HPV-associated pathology are difficult to obtain as the virus life cycle depends on a proper epithelial differentiation. Nevertheless, compelling evidences have shown that the E7 early gene from HPV 16 binds and induces protein instability of the pocket family of proteins pRb, p107 and p130, thus contributing essentially to HPV-oncogenesis. The major aims of this project are to characterize the capability of the E7 gene of HPV 5 to induce instability of pocket proteins, and to determine whether human primary keratinocytes transduced with the E7 genes from HPV 5 or HPV 16 (5E7 and 16E7), and transplanted into inmunodeficient mice as reconstituted skin, could be used as models of HPV-pathobiology. The results that we have obtained demonstrate that 5E7 is able to bind pRb in vitro. Moreover, 5E7 reduce the protein levels of pRb and p107 similarly to 16E7, and in a manner that is dependant on an intact LxCxE pocket binding motif. On the other hand, transplants with 5E7 or 16E7 genes demonstrated stable transgene expression during the analysis period (3 to 6 months). Histological examination of 16E7 epidermis showed similarities with pretumoral intrapithelial neoplasia of the cervix (acanthosis, suprabasal mitosis, papillomatosis, hyperkeratosis or nuclear atypia) which were not present in 5E7 skin. Moreover, transplants expressing 16E7 display disrupted differentiation. Suprabasal proliferation and cell cycle reentry was frequently observed in 16E7 transplants but focally in 5E7 samples. Molecular analysis of surrogate biomarkers of high-risk HPV-infected tumors demonstrated that expression of the E7 protein in reconstituted human skin transplanted into immunodeficient mice is a good in vivo model of HPV pretumoral and tumoral lesions. Moreover, this model could be used to analyze preclinical new antitumor therapies.