Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21WAF1 and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.

Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of integral membrane transporters, such as P-glycoprotein (Pgp) and MDR-associated proteins (MRPs), with resulting decrease of drug accumulation and cellular death. Doxorubicin is a substrate of Pgp; it has been suggested that its ability to induce synthesis of nitric oxide (NO) could explain, at least in part, its cytotoxic effects. Culturing the human epithelial colon cell line HT29 in the presence of doxorubicin, we obtained a doxorubicin-resistant (HT29-dx) cell population: these cells accumulated less intracellular doxorubicin, were less sensitive to the cytotoxic effects of doxorubicin and cisplatin, overexpressed Pgp and MRP3, and exhibited a lower NO production (both under basal conditions and after doxorubicin stimulation). The resistance to doxorubicin could be reversed when HT29-dx cells were incubated with inducers of NO synthesis (cytokines mix, atorvastatin). Some NO donors increased the drug accumulation in HT29-dx cells in a guarosine-3′:5′-cyclic monophosphate–independent way; this effect was associated with a marked reduction of doxorubicin efflux rate in HT29 and HT29-dx cells, and tyrosine nitration in the MRP3 protein. Our results suggest that onset of MDR and impairment of NO synthesis are related; this finding could point to a new strategy to reverse doxorubicin resistance in human cancer.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.