Interaction of human papillomavirus 20 and 4-nitroquinoline-1-oxide in carcinogenesis

Abstract

Human papillomaviruses (HPV) infect mucosal and cutaneous tissue. They induce several epithelial lesions ranging from benign warts to cancer. HPV infection has been demonstrated to play a role in the pathogenesis of cervical, vulvar, penile, anal, skin, head and neck cancers. HPV infection is associated with benign and malignant lesions in the hereditary skin disease Epidermodysplasia verruciformis (EV), characterized by cutaneous lesions that progress to squamous cell carcinoma after UV exposure. Head and neck tumor (HNSCC) patients have been classified into two epidemiological entities, corresponding to either alcohol/tobacco use or HPV infection. High risk mucosal HPV is involved in the pathogenesis of a subset of HNSCC, particularly those that arise from the lingual and palatine tonsils within the oropharynx. Although low risk HPV have been found in several cancers, their gene products do not induce immortalization per se, and they probably need other co-factors to modify cellular genes and contribute to tumorigenesis. Cutaneous HPV20 has been found in a number of non melanoma skin cancers as well as in esophageal carcinoma and in HNSCC patients. HPV20 infection combined with UV exposure promotes malignant progression in skin as demonstrated in HPV20 E6/E7 transgenic mice in the presence of chronic UV irradiation. These animals presented papilloma formation that lead to transformation. However, involvement of HPV20 in esophageal cancer and HNSCC have to date not been elucidated. In this study, the cooperation between HPV20 infection and a chemical carcinogen in the pathogenesis of oropharynx and esophageal cancer was investigated. The synthetic carcinogen 4-NQO is a UV-mimetic that shares the same mechanistic action with the carcinogenic compounds present in tobacco. In order to examine its involvement (comparable with the effect of tobacco) in increasing the viral oncogenic potential, 4-NQO was applied either to cells or organotypic cultures and combined with HPV20 by following two approaches: Promoter activation assays demonstrated that HPV20 E6 and/or E7 up-regulated its own viral promoter and this effect was further enhanced by 4-NQO treatment. These changes in the viral promoter activity may indicate a positive cooperation between the virus and the chemical in the onset of disease. Secondly, alterations in proliferation, differentiation, apoptosis and skin barrier functionality were studied in organotypic cultures expressing HPV20 E6 and treated with 4-NQO. Although these cultures were able to stratify and differentiate they failed to form a normal epidermis. Moreover, the normal differentiation markers pattern was disturbed, resembling simpler epithelia and this phenotype was enhanced after 4-NQO treatment. Cellular protein p53 accumulated whereas p21CIP1 and ΔNp63α modifed their expression pattern in the epithelia. Organotypic raft cultures expressing HPV20 E6 gene presented foamy-like keratinocytes resembling the typical structures found in Epidermodysplasia verruciformis patients and pityriasis versicolor -like lesions infected with HPV20. In addition, HPV20 E6 expression was capable of altering the functionality of the skin barrier function by abrogation of tight junction formation in the stratum granulosum and by changes in the lipid metabolism of the skin leading to its accumulation and deregulated composition. Disruption of the skin barrier has been observed in a number of cutaneous diseases as ichthyosis, atopic dermatitis and other eczemas. These studies provide insight into the functionality of HPV20 E6 in the epidermis. Moreover, the up-regulation in viral promoter activity and alterations in differentiation pattern under the context of HPV20 E6 expression and 4-NQO treatment compared with pLXSN and HPV16 E6/E7 controls showed the cooperation between HPV20 and chemical carcinogens. However, epithelial transformation was not observed and longer 4-NQO treatment would be necessary to analyze its influence in malignancy. Taken together, the conclusions obtained in the present work will be helpful in future experiments to elucidate the role of HPV20 infection and chemical carcinogens in the pathogenesis of esophageal and oropharyngeal tumors.